AU2014301147B2 - Modified diatoms for biofuel production - Google Patents

Abstract

The invention provides engineered diatoms and methods of producing oil using diatoms. The invention also provides methods of modifying the lipids quantity and/or quality produced by diatom organisms through genome engineering. Also provided are oils, fuels, oleochemicals, chemical precursors, and other compounds manufactured from such modified diatoms.

Description

MODIFIED DIATOMS FOR BIOFUEL PRODUCTION FIELD OF THE INVENTION

The invention provides engineered diatoms and methods of producing oil using diatoms. The invention also provides methods of modifying the lipids quantity and/or quality

produced by diatom organisms through genome engineering. Also provided are oils, fuels,

oleochemicals, chemical precursors, and other compounds manufactured from such modified diatoms.

BACKGROUND OF THE INVENTION

Concerns about rises in prices of fossil fuels have prompted intense interest in the development of engineered microorganisms as attractive sources for the production of

biofuel. Photosynthetic algae have been of considerable interest as a possible biofuel

resource for decades. Diatoms are one of the most ecologically successful unicellular phytoplankton on the planet, being responsible for approximately 20% of global carbon fixation, representing a major participant in the marine food web. They are able to

accumulate abundant amounts of lipid suitable for conversion to liquid fuels and because

of their high potential to produce large quantities and varieties of lipids and good growth efficiencies, they are considered as one of the best classes of algae for renewable biofuel

production (Kroth 2007; Saade and Bowler 2009).

Nevertheless, relatively little is known about lipid metabolism in these algae. Extensive

knowledge on complex lipid metabolism pathways is gained mostly from studies of plant or animal models. Genetic engineering of diatoms lipid gene is indispensable to

understand the complex lipid metabolism and improve lipid production. However, despite the recent publication of Thalassiosira pseudonana (Armbrust, Berges et al. 2004) and Phaeodactylum tricornutum genomes (Bowler, Allen et al. 2008), very few genetic tools

to explore diatoms genetics are available at this time: annotations of the diatoms

genomes remain essentially based on putative open reading frames without confirmation of actual gene function. For instance, the direct manipulation of target genes by homologous recombination has proven difficult and the generation of loss of function mutants by insertional or chemical mutagenesis is challenging in diatoms because they are diploid organisms. This considerably limits the use of these organisms for biofuel applications. One genetic engineering study has succeeded to increase the amount of lipid within diatom.

However, this was made by random integration of two transgenes involved in lipid metabolism (Radakovits, Eduafo et al. 2011).

SUMMARY OF THE INVENTION

Based on genome comparison and protein homology search, the inventors selected several

target genes involved in lipid metabolism and, for the first time, selectively inactivated them in order to create new diatom strains for biofuel production. Generation of modified diatoms was

facilitated by using specific rare-cutting endonuclease, in particular TAL-nucleases, MBBBD nucleases and/or CRISPR/Cas9-nucleases, allowing specific gene targeting within the diatom

genome. The inventors thereby generated diatoms in which inactivation of the selected genes induces an increase quantity and/or quality of lipid content.

According to a first aspect, the present invention provides a method for producing lipids

comprising the step of:

(a) cultivating in a culture medium a diatom strain in which a gene selected from elongase and UDP-glucose pyrophosphorylase genes has been inactivated by a rare-cutting

endonuclease selected from a TALE-nuclease, a MBBBD-nuclease and a CRISPR/Cas9 nuclease;

(b) harvesting said cultivated diatom strain;

(c) extracting the lipids from said harvested diatoms.

According to a second aspect, the present invention provides a method according to the first

aspect further comprising the step of producing biofuel from the extracted lipids.

(24641715_1):GGG

2a

According to a third aspect, the present invention provides a method according to the first

aspect further comprising the step of transforming the extracted lipids into a cosmetic or a food product.

According to a fourth aspect, the present invention provides lipids prepared according to the method of the first aspect.

According to a fifth aspect, the present invention provides biofuel prepared according to the method of the second aspect.

According to sixth aspect, the present invention provides a cosmetic or food product prepared

according to the method of the third aspect.

BRIEF DESCRIPTION OF FIGURES Figure 1: Starch metabolism in green microalgae. Glucans are added to the water soluble

polysaccharide (WSP) by a-1,4 glycosidic linkages (WSP1) until a branching enzyme highly branches the ends (WSP2). Some of these branches are trimmed (WSP3), and this process is

repeated until a starch granule is formed. Phosphorolytic [Starch-(P)n] and hydrolytic degradation pathways are shown. aAMY, a-amylase; AGPase, ADP-glucose pyrophosphorylase;

pAMY, -amylases; BE, branching enzymes; DBE, debranching enzymes; DPE, disproportionating enzyme (1 and 2)a-1,4 glucanotransferase; Glc, glucose; GWD, glucan-waterdikinases; ISA, isoamylases; MEX1, maltose transporter; MOS, malto-oligosaccharides; PGM, plastidial

phosphoglucomutase; P, phosphate; Pi,,

(24641715_1):GGG inorganic phosphate; PPi, pyrophosphate; SP, starch phosphorylases; SS, starch synthases. (Radakovits, Jinkerson et al. 2010)

Figure 2: Representative pathways of microalgal lipid biosynthesis. Free fatty acids are

synthesized in the chloroplast, while TAGs may be assembled at the ER. ACCase, acetyl CoA carboxylase; ACP, acyl carrier protein; CoA, coenzyme A; DAGAT, diacylglycerol

acyltransferase; DHAP, dihydroxyacetone phosphate; ENR, enoyl-ACP reductase; FAT,

fatty acyl-ACP thioesterase; G3PDH, gycerol-3-phosphate dehydrogenase; GPAT, glycerol 3-phosphate acyltransferase; HD, 3-hydroxyacyl-ACP dehydratase; KAR, 3-ketoacyl-ACP

reductase; KAS, 3-ketoacyl-ACP synthase; LPAAT, lyso-phosphatidic acid acyltransferase; LPAT, lyso-phosphatidylcholine acyltransferase; MAT, malonyl-CoA:ACP transacylase;

PDH, pyruvate dehydrogenase complex; TAG, triacylglycerols. (Radakovits, Eduafo et al. 2011)

Figure 3: Possible biosynthetic routes leading to eicosapentaenoic acid (EPA) biosynthesis in Phaeodactylum tricornutum. The classical w6- and w3-pathways are framed and the

alternative w3-pathway (involving A9-elongation and A8-desaturation) is shown with broken arrows. (Domergue, Lerchl et al. 2002)

Figure 4: Molecular characterization of clones from the transformation of the Phaeodactylum

tricornutum (Pt) strain with the TALE-Nuclease targeting the UGPase gene. Amplification of the UGPase locus by PCR surrounding the TALE-Nuclease cleavage site and migration of the PCR products on agarose gel. Four clones presented a PCR product with a higher size than the one expected (37-5A3, 37-7A1, 37-7B2 and 37-16A1), one clone was not amplified (37-8A1) and 7 presented a PCR band at the expected size as observed in the two clones from the transformation with the empty vector (37-3B1 and 37-3B2).

Figure 5: Molecular characterization of clones from the transformation of the Phaeodactylum tricornutum (Pt) strain with the TALE-Nuclease targeting the UGPase gene (experiment 1). T7 assay performed on the 12 clones from the transformation with UGP_TALE-Nuclease and 2 clones from the transformation with the empty vector. The negative control corresponds to a PCR carried out on the clone 37-3B1 (transformed with the empty plasmid), not digested by the T7 enzyme. The T7 positive control corresponds to a PCR product carrying mutagenic events. The clone 37-5B4 is positive for T7 assay.

Figure 6: Molecular characterization of clones from the transformation of the

Phaeodactylum tricornutum (Pt) strain with the TALE-Nuclease targeting the UGPase gene (experiment 2). (A) Amplification of the UGPase locus by PCR surrounding the TALE

Nuclease cleavage site and migration of the PCR products on an agarose gel. On the 11 clones tested, five were not amplified by PCR (42-5A2, 42-5A6, 42-6B2, 42-8B1 and 42

7A7). The other clones presented a PCR product at the expected size. The clones 42-3B1 and 42-3B2 correspond to controls resulting from the transformation with the empty

vector. (B) T7 assay performed on the 6 clones from the transformation with the UGPTALE-Nuclease and 2 clones from the transformation with the empty vector. The

negative control corresponds to a PCR performed on the clone 37-3B1 (transformed with

the empty plasmid), not digested by the T7 enzyme. The T7 positive control corresponds to a PCR product carrying mutagenic events. The clones 42-5A1, 42-6B5, 42-7A2 and 42 7A3 are positive for T7 assay.

Figure 7: Example of a mutagenic event induced by the TALE-Nuclease targeting the UDP glucose pyrophosphorylase gene (UGPase).

Figure 8: Molecular characterization of clones from the transformation of the Pt strain with the TALE-Nuclease targeting the UGPase gene. Clone 37-7A1: 100% mutated on the UGPase gene, clone 37-3B1 from transformation with the empty vector and the Pt wild

type strain were labeled with the lipid probe (Bodipy (493/503), Molecular Probe). The

fluorescence intensity was measured by flow cytometry. The graphs represent the number of cells function of the fluorescence intensity for 3 independent experiments.

Figure 9: Quantitative analysis of the fatty acid (FA) and the triacylglycerol (TAG) content in the transgenic diatoms strain corresponding to the mutant UGPase KO (37-7A1) and its

associated controls empty vector and Pt wild type.

Figure 10: Mutagenesis induced by the TALE-Nuclease targeting the putative elongase

gene. A PCR surrounding the putative elongase specific target was performed. In the left panel, the clone presenting in equal proportions a PCR band at the expected size and another one with a higher size discloses a clear mutagenic event. A T7 assay was assessed on 4 clones resulting from the transformation with the elongase TALE-Nuclease and on 3 clones resulting from the transformation with the empty vector. The clone 2 is positive for the T7 assay.

Figure 11: Example of a mutagenic event induced by the TALE-Nuclease targeting the

elongase gene.

Figure 12: Quantitative analysis of the fatty acid (FA) and the triacylglycerol (TAG) content in the transgenic diatoms strain corresponding to the mutant Elongase and its associated

control empty vector.

Figure 13: Example of a mutagenic event induced by TALE-Nuclease within endogenous

Glycerol 3 Phosphate deshydrogenase (G3PDH).

Figure 14: Example of a mutagenic event induced by TALE-Nuclease within endogenous omega 3 desaturase gene.

Figure 15: Example of a mutagenic event induced by TALE-Nuclease within endogenous

palmitoyl protein thioesterase gene.

Figure 16: Example of a mutagenic event induced by TALE-Nuclease within endogenous

Enoyl ACP reductase gene.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy,

biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in

the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated,

conventional techniques of cell biology, cell culture, molecular biology, transgenic

biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current

Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195;

Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. 1. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A

Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.

Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically,

Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And

Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of

Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, N.Y., 1986).The present invention relates to a modified diatom strain with high lipid quantity and/or quality content especially for biofuel production. In particular, the

present invention relates to a modified diatom strain in which a gene involved in lipid

metabolism has been inactivated. By inactivated, it is meant, that the gene encodes a non-functional protein or does not express the protein. Said gene is preferably inactivated

by a rare-cutting endonuclease, more preferably by a TALE-nuclease, a MBBD-nuclease or a CRISPR/Cas9 nuclease.

Another option for gene inactivation is the use of RNA silencing to knock down gene expression (De Riso, Raniello et al. 2009) and particularly small-hairpin RNA (shRNA) that

target nucleic acid encoding protein involved in lipid metabolism. Recent improvements in gene knockdown strategies include the development of high-throughput artificial micro-RNA (armiRNA) techniques that are reportedly more specific and stable (Molnar, Bassett et al. 2009; Zhao, Wang et al. 2009). Another inactivation tool can be a double

strand DNA, repressor molecules or dominant negative inhibitor protein capable of interrupting protein expression or function.

As a result, inactivation of said gene induces the production of an increased amount, storage and/or quality of lipids in diatom.

Diatoms are unicellular phototrophs identified by their species-specific morphology of

their amorphous silica cell wall, which vary from each other at the nanometer scale.

Diatoms includes as non limiting examples: Phaeodactylum, Fragilariopsis, Thalassiosira, Coscinodiscus, Arachnoidiscusm, Aster omphalus, Navicula, Chaetoceros, Chorethron, Cylindrotheca fusiformis, Cyclotella, Lampriscus, Gyrosigma, Achnanthes, Cocconeis,

Nitzschia, Amphora, and Odontella.

In a more preferred embodiment, diatoms according to the invention are from the

species: Thalassiosira pseudonana or Phaeodactylum tricornutum.

By "genes involved in lipid metabolism" is meant any putative gene from the diatoms genomes that has similarity with a gene characterized in the literature encoding a protein

taking part one biochemical reactions of lipid biosynthesis and catabolism, in particular

one of the pathways illustrated in figures 1, 2 or 3, as well as pathways that modify the length and/or saturation of fatty acids (see for review, (Radakovits, Jinkerson et al. 2010).

The invention envisions that many genes involved in lipid biosynthesis can be subjected to knock-out or knock-in, individually or collectively, in order to increase the production or

storage (internal accumulation) of lipids and/or to improve the quality of the lipids.

The genes encoding enzymes involved in the pathways of fatty acid synthesis can encode proteins having for instance acetyl-CoA carboxylase, fatty acid synthase, 3-ketoacylacyl- carrier protein synthase Il, glycerol-3-phospate deshydrogenase (G3PDH), Enoyl-acyl carrier protein reductase (Enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase, lysophosphatidic acyl transferase or diacylglycerol acyltransferase, phospholipid:diacylglycerol acyltransferase, phoshatidate phosphatase, fatty acid thioesterase such as palmitoyl protein thioesterase, or malic enzyme activities (see figure 2).

Another strategy to induce lipid accumulation within diatom is to decrease lipid catabolism. Genes involved in the activation of both triacylglycerol and free fatty acids, as

well as genes directly involved in p-oxidation of fatty acids can be inactivated to increase cellular lipid content. For example, acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA

oxidase activity, phosphoglucomutase, can be inactivated. Lipases are enzymes that de esterify carboxyl esters, such as triacylglycerols and phospholipids. Many of putative

lipase can be found in diatoms. As non limiting example in P. tricornutum Phatrdraft_44231 which encodes a putative tricaylglycerol lipase, Phatrdraft_50397 can

be inactivated to induce lipid accumulation.

According to the invention, the cellular lipid content of the diatoms can also be increased

by inactivating metabolic pathways leading to the accumulation of energy-rich storage compounds, such as chrysolaminarin (P-1, 3-glucan). For instance, UDP-glucose

pyrophosphorylase, ADP-glucose pyrophosphorylase, isoamylase genes can be inactivated in these diatoms strains (see figure 1).

In addition, the quality of lipids can be increased with regard to suitability as biofuel, by modifying genes involved in the carbon chain length and the degree of unsaturation of

the fatty acids which can affect the cold flow and oxidative stability properties of the biofuel derived from the feedstock of the diatom. Examples of these are delta 12 desaturase, delta 9 desaturase, omega 3 desaturase and elongase enzymes (see figure 3).

In another aspect, as non-limiting examples, thioesterases enzymes, such as acyl-ACP

thioesterases specific for shorter chain length fatty acids can be overexpressed to improve cold flow properties (Hu, Sommerfeld et al. 2008; Radakovits, Jinkerson et al.

2010).

Here, the present inventors have more particularly identified a selection of putative genes in the diatoms genomes encoding putative enzymes selected from the group consisting

of: glycerol-3-phosphate deshydrogenase, w3-desaturase, palmitoyl protein thioesterase,

Enoyl ACP reductase, A12 desaturase, UDP-glucose pyrophosphorylase and elongase.

They have designed rare-cutting endonuclease capable of targeting these genes or gene

sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% sequence identity with any one of the sequences selected from the group consisting of: SEQ ID NO:

3, 14, 22, 30, 36, 42 and 48. In particular embodiment, the rare-cutting endonuclease is capable of cleaving target sequence selected from the group consisting of: SEQ ID NO: 6,

17,25,33,39,45 and 51.

The resulting diatoms modified according to the invention can produce an increased

amount of lipid per cell of at least 10% compared to the wild type strain, particularly at least 20, 30, 40, 50 %, more preferably at least 75%, 100%, 200%, 300% compared to the

wild type strain. In another words, the present invention relates to modified diatoms with a lipid content of at least 30 %, preferably at least 40%, 50%, 60%, 70%, 80,% of dry

weight.

In particular embodiment, the present invention relates to modified diatoms which

preferably produce an increased amount of shorter chain length fatty acids compared to wild type, preferably fatty acids having chain of 12, 14, 16, 18, 20 carbons, preferably between 16 and 20, more preferably between 16 and 18 carbons, again more preferably

between 12 and 16 carbons. In another particular embodiment, the present invention

relates to modified diatoms which preferably produce fatty acids with a lower degree of unsaturation, preferably modified diatoms produce an increase amount of fatty acids

with no more than 5 preferably 4, 3, 2 or 1 double bond(s) between carbon atoms, more preferably fatty acids with no double bonds between carbon atoms (saturated fatty

acids).

By increased amount of product, it is meant that the modified diatoms present an

increase production of at least 10%, preferably of at least 20%, 30%, 40% or 50 %, more preferably at least 75%, 100%, 200%, 300% compared to the wild type strain.

The lipid content analysis can be performed following protocol previously described in (Vieler, Wilhelm et al. 2007; Lamaziere, Wolf et al. 2012; Lamaziere, Wolf et al. 2013).

Due to the ease of the present genetic engineering method, modified diatom strains can

comprise simultaneous modifications to modulate the lipid metabolic pathway, for instance simultaneous activation and/or inactivation of several key enzymes involved in

lipid metabolism.

In particular embodiment, the modified diatoms can comprise one inactivated lipid gene

by insertion of a transgene. In particular embodiment, said transgene encodes for an enzyme involved in the lipid metabolism. In this case, both inactivation of endogenous

gene and overexpression of the transgene can improve the production and the quality of lipid within diatoms. As non-limiting example, said modified diatom can comprise an

inactivated gene selected from the group consisting of: glycerol-3-phosphate

deshydrogenase, w3-desaturase, palmitoyl protein thioesterase, eonyl ACP reductase,

elongase, UDP-glucose pyrophosphorylase and A12 desaturase genes, and a transgene comprising at least one thioesterase gene.

The lipid gene according to the present invention can be modified by introducing into the diatom a DNA binding domain which specifically targets the lipid gene of interest. In

particular, the method for lipid gene targeted modification in diatom can comprise: selecting a target sequence within a gene of a diatom strain putatively involved in lipid metabolism; providing a DNA binding domain to target said gene; introducing said DNA

binding domain into diatom; optionally selecting diatom producing an increased amount,

storage and/or quality of lipids. Said DNA binding domain can be as non limiting examples a TALE binding domain or a MBBBD binding domain. Said DNA binding domain can be

fused with a transcription activator or a repressor (i. e. a transcription regulator) or a

protein that interacts with or modifies other proteins implicating in DNA processing. Non limiting examples of DNA processing activities can be for example creating or modifying

epigenetic regulatory elements, making site-specific insertions, deletions, or repairs in DNA, controlling gene expression, and modifying chromatin structure.

In a particular aspect of the invention, the lipid gene according to the present invention can be modified by introducing into the diatom a rare-cutting endonuclease which

specifically cleaves the lipid gene of interest. In particular, the method for lipid gene targeted modification in diatom can comprise: selecting a target sequence within a gene

of a diatom strain putatively involved in lipid metabolism; providing a rare-cutting endonuclease to target and inactivate said gene; introducing said rare-cutting

endonuclease into diatom; optionally selecting diatom in which said putative gene involved in lipid metabolism has been inactivated and producing an increased amount,

storage and/or quality of lipids. Said rare-cutting endonuclease can be as non-limiting

example, a TALE-nuclease, a MBBBD-nuclease or a CRISPR/Cas9 nuclease which is capable of targeting specifically the selected target sequence. Preferably, selected target

sequence is comprised within a putative gene involved in the lipid metabolism as described above. In particular, said target sequence is comprised within a gene selected

from the group consisting of: glycerol-3-phosphate deshydrogenase, w3-desaturase, palmitoyl protein thioesterase, eonyl ACP reductase, elongase, UDP-glucose

pyrophosphorylase and A12 desaturase genes. More particularly, said rare-cutting endonuclease is capable of targeting a gene having at least 70%, preferably at least 75%,

80%, 85%, 90%, 95% sequence identity with any one of the sequences selected from the group consisting of: SEQ ID NO: 3, 14, 22, 30, 36, 42 and 48. In particular embodiment,

the rare-cutting endonuclease is capable of cleaving target sequence selected from the group consisting of: SEQ ID NO: 6, 17, 25, 33, 39, 45 and 51. By "cleavage", it is meant a

double strand break or single strand break in the target sequence. It is also encompassed in the present invention said TALE-nucleases, preferably said TALE-nuclease encoding by

the plasmid sequence selected from the group consisting of: SEQ ID NO: 4, 5, 15, 16, 23, 24,31,32,37,38,43,44,49and50.

Said modified target sequence can result from NHEJ events or homologous recombination. The double strand breaks caused by said rare-cutting endonucleases are

commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). Although homologous recombination typically uses the sister chromatid of the damaged DNA as a donor matrix from which to perform perfect repair of the genetic lesion, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the double strand break. Mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (Ma, Kim et al. 2003). Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used for the creation of specific gene knockouts.

In a particular embodiment of the methods envisaged herein the mutagenesis is increased by transfecting the cell with a further transgene coding for a catalytic domain.

In a more preferred embodiment, said catalytic domain is a DNA end-processing enzyme. Non limiting examples of DNA end-processing enzymes include 5-3' exonucleases, 3-5'

exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, hosphatase, hydrolases and template-independent DNA polymerases. Non limiting examples of such

catalytic domain comprise a protein domain or catalytically active derivate of the protein domain selected from the group consisting of hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase) Human DNA2, Yeast DNA2

(DNA2_YEAST). In a more preferred embodiment, said catalytic domain has an

exonuclease activity, in particular a 3'-5' exonuclease activity. In a more preferred embodiment, said catalytic domain has TREX exonuclease activity, more preferably TREX2

activity. In another preferred embodiment, said catalytic domain is encoded by a single chain TREX polypeptide. In a particular embodiment, said catalytic domain is fused to the

N-terminus or C-terminus of said rare-cutting endonuclease. It has been found that the coupling of the enzyme SCTREX2 with an endonuclease such as a TALE-nuclease ensures

high frequency of targeted mutagenesis (W02012054858, W02013009525).

Endonucleolytic breaks are known to stimulate homologous recombination. Therefore, in

particular embodiments, said modified target sequence can result to donor matrix insertion (knock-in) into chosen loci of the genome. In particular embodiments, the

knock-in diatom is made by introducing into said diatom a genome engineering nuclease as described above, to induce a cleavage within or adjacent to target sequence, and a donor matrix comprising a transgene to introduce said transgene by a knock-in event. Said donor matrix comprises a sequence homologous to at least a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target DNA sequence and the donor matrix. In particular embodiments, said donor matrix comprises first and second portions which are homologous to region 5' and 3' of the target nucleic acid, respectively. Following cleavage of the target nucleic acid sequence, a homologous recombination event is stimulated between the genome containing the target nucleic acid sequence and the donor matrix. Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used within said donor matrix. Therefore, the donor matrix is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp.

Depending on the location of the targeted sequence wherein cleavage event has

occurred, such donor matrix can be used to knock-out a gene, e.g. when the donor matrix is located within the open reading frame of said gene, or to introduce new sequences or

genes of interest. Sequence insertions by using such donor matrix can be used to modify a targeted existing gene, by correction or replacement of said gene (allele swap as a non

limiting example), or to up- or down-regulate the expression of the targeted gene

(promoter swap as non-limiting example), said targeted gene correction or replacement conferring one or several commercially desirable traits.

In particular embodiment, said donor matrix can comprise a transgene encoding an

enzyme involved in the lipid metabolism. Said donor matrix can be inserted in the target sequence by homologous recombination. The transgene replaces and inactivates the

target gene. In this case, both inactivation of endogenous gene and overexpression of the

transgene can improve the production and the quality of lipid within diatoms. As non limiting example, said donor matrix can comprise a thioesterase gene and the target

sequence can be selected from the group consisting of: glycerol-3-phosphate deshydrogenase, w3-desaturase, palmitoyl protein thioesterase, Enoyl ACP reductase,

A12 desaturase, UDP-glucose pyrophosphorylase and elongase genes.

Molecules can be introduced into the diatom by transformation method well-known in the art. In various embodiments, nucleotide sequence for example vector encoding rare

cutting endonuclease and/or donor matrix can be introduced into diatom nuclei by for example without limitation, electroporation, magnetophoresis, micropartile bombardment. Direct introduction of purified endonucleases of the present invention in

diatom can be considered.

Transformation methods require effective selection markers to discriminate successful transformants cells. The majority of the selectable markers include genes with a

resistance to antibiotics. Only few publications refer to selection markers usable in Diatoms. (Dunahay, Jarvis et al. 1995) report the use of the neomycin phosphotransferase

II (nptll), which inactivates G418 by phosphorylation, in Cyclotella cryptica, Navicula saprophila and Phaeodactylum tricornutum species. (Falciatore, Casotti et al. 1999;

Zaslavskaia, Lippmeier et al. 2001) report the use of the Zeocin or Phleomycin resistance gene (Sh ble), acting by stochiometric binding, in Phaeodactylum tricornutum and Cylindrothecafusformis species. In (Falciatore, Casotti et al. 1999; Zaslavskaia, Lippmeier

et al. 2001), the use of N-acetyltransferase 1 gene (Nat1) conferring the resistance to

Nourseothricin by enzymatic acetylation is reported in Phaeodactylum tricornutum and

Thalassiosira pseudonana. It is understood that use of the previous specific selectable markers are comprised in the scope of the present invention and that use of other genes encoding other selectable markers including, for example and without limitation, genes

that participate in antibiotic resistance. In a more preferred embodiment, the vector

encoding for selectable marker and the vector encoding for rare-cutting endonuclease are different vectors.

Increase lipid synthesis can result in a reduction of cell division. In such case, modification of lipid gene expression can be beneficial if they can be controlled by an inducible

promoter that can be activated for instance once the modified diatoms have grown to a high density and have entered stationary phase. Thus, in particular embodiments, the

gene encoding a rare-cutting endonuclease or the transgene according to the present invention can be placed under the control of a promoter. An inducible promoter is a promoter which initiates transcription only when it is exposed to some particular (typically external) stimulus. Particularly preferred for the present invention are: a light regulated promoter, nitrate reductase promoter, eukaryotic metallothionine promoter, which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-p-D-thiogalacto-pyranoside (IPTG), steroid-responsive promoter, tetracycline-dependent promoter and eukaryotic heat shock promoter which is induced by increased temperature.

In another aspect, it is also encompassed in the scope of the present invention, a

modified algal cell obtained or obtainable by the methods described above. In particular embodiments, such modified algal cells are characterized by the presence of a sequence

encoding a rare-cutting endonuclease transgene and a modification in a targeted lipid gene, preferably in both alleles.

The present invention also relates to methods to produce biofuel using the modified diatoms described above.

In particular, the present invention relates to a method for producing lipids comprising

one or severalofthe steps of:

(a) cultivating a modified diatom strain as described above in a adapted culture medium,

(b) optionally, harvesting modified diatom strains,

(c) extracting the lipids from the diatoms.

Several extraction methods for lipids are well-known in the art: physical extraction, chemical extraction, supercritical fluid extraction, in situ extraction, ultrasonic assisted

extraction or pulsed electric field technology. Physical methods destruct the algal cells and consist of sonication, homogenization, French pressing, expelling and beads milling.

For the chemical solvent extraction, several extractors and mixtures are known; for example, hexane, chloroform, methanol, isopropanol and acetone. For the supercritical

fluid extraction, the extraction medium is in many cases C02. In the in situ extraction, the

algae are not harvested and do not need to bedewatered or dried. The lipids are extracted from living cells (Frenz, Largeau et al. 1989; King 1996; Lee, Yoon et al. 1998; Sievers 1998; Hejazi and Wijffels 2004; Herrero, Jaime et al. 2006; Doucha and Livansky

2008; Wei, Gao et al. 2008; Shen, Yuan et al. 2009; Mercer and Armenta 2011).

The present invention also relates to a method comprising the step of producing biofuel from the lipids produced by diatoms, especially triacylglycerol compounds.

The biofuel production can be performed as described in (Kr6ger and MOller-Langer 2012), W02009063296). The biofuel production can be realized via (trans)esterification,

in situ transesterification wherein the algae medium is directly mixed with the solvent, catalyst and alcohol, by hydroprocessing from algal lipids called hydroprocessed esters

and fatty acids. The present invention also relates to a step of transforming the extracted lipids into a cosmetic or a food product, especially for their high content of essential fatty acids, more particularly as containing omega-3 fatty acids, such as docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA or icosapentaenoic acid).The present invention also encompasses other uses of the modified diatoms or their extracted lipids. In particular, the modified diatoms

according to the invention can be cultivated for their oil contents and directly used under

their algal forms, as an essential source of fatty acids in animal alimentation, in particular to breed fish or shellfish.

Definitions:

By "gene" it is meant the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of

protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns and a 3' untranslated region. The gene may

further be comprised of terminators, enhancers and/or silencers.

By "genome" it is meant the entire genetic material contained in a cell such as nuclear

genome, chloroplastic genome, mitochondrial genome.

As used herein, the term "locus" is the specific physical location of a DNA sequence (e.g. of a gene) on a nuclear, mitochondria or choloroplast genome. As used in this

specification, the term "locus" usually refers to the specific physical location of an endonuclease's target sequence. Such a locus, which comprises a target sequence that is recognized and cleaved by an endonuclease according to the invention, is referred to as

"locus according to the invention".

By "target sequence" is intended a polynucleotide sequence that can be processed by a rare-cutting endonuclease according to the present invention. These terms refer to a

specific DNA location, preferably a genomic location in a cell, but also a portion of genetic

material that can exist independently to the main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria or

chloroplasts as non-limiting examples. The nucleic acid target sequence is defined by the 5' to 3' sequence of one strand of said target.

As used herein, the term "transgene" refers to a sequence inserted at in an algal genome.

Preferably, it refers to a sequence encoding a polypeptide. Preferably, the polypeptide

encoded by the transgene is either not expressed, or expressed but not biologically active, in the diatom in which the transgene is inserted. Most preferably, the transgene encodes a polypeptide useful for increasing the quantity and/or the quality of the lipid in

the diatom. Also, the transgene can be a sequence inserted in an algae genome for

producing an interfering RNA.

By "homologous" it is meant a sequence with enough identity to another one to lead to homologous recombination between sequences, more particularly having at least 95% identity, preferably 97% identity and more preferably 99%.

"Identity" refers to sequence identity between two nucleic acid molecules or

polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared

sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a

function of the number of identical or matching nucleotides at positions shared by the

nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting.

By "DNA binding domain", it is meant a protein domain capable of binding a target nucleic

acid sequence, preferably a DNA molecule. The DNA binding domain recognizes and binds nucleic acid at specific polynucleotide sequences, further referred to as "nucleic acid

target sequence". Transcription Activator like Effector (TALE) are proteins from the

bacterial species Xanthomonas comprise a plurality of repeat sequences, each repeat comprising di-residues in position 12 and 13 (RVD) that are specific to each nucleotide

base of the nucleic acid targeted sequence. TALE binding domain is composed by a variable number of 33-35 amino acid repeat modules. These repeat modules are nearly

identical to each other except for two variable amino acids located at positions 12 and 13 (i.e. Repeat Variable Di residues, RVD). The nature of residues 12 and 13 determines base

preferences of individual repeat module. Preferably, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for

recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for

recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and

YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A. In another embodiment, critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards

nucleotides A, T, C and G and in particular to enhance this specificity. Binding domains

with similar base-per-base nucleic acid binding properties (modular base-per-base specific nucleic acid binding domains (MBBBD) can also be derived from new modular proteins

recently discovered by the applicant in a different bacterial species. Said MBBBD can be engineered, for instance, from the newly identified proteins, namely EAV36_BURRH,

E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH proteins from the recently

sequenced genome of the endosymbiont fungi Burkholderia Rhizoxinica (Lackner, Moebius et al. 2011).

By "rare-cutting endonuclease",it is meant any wild type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA

molecule, preferably a DNA molecule.A rare-cutting endonucelase is highly specific, recognizing nucleic acid target sites ranging from 10 to 45 base pairs (bp) in length, usually ranging from 10 to 35 base pairs in length. . The endonuclease according to the

present invention recognizes and cleaves nucleic acid at specific polynucleotide

sequences, further referred to as "nucleic acid target sequence".

"TALE-nuclease" or "MBBBD-nuclease" refers to engineered proteins resulting from the

fusion of a nucleic acid binding domain typically derived from Transcription Activator like Effector proteins (TALE) or MBBBD binding domain, with an endonuclease catalytic

domain. Such catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, like for instance I-Tev, CoIE7, NucA and Fok-l. In a

more preferred embodiment, said nuclease is a monomeric TALE-Nuclease or MBBBD nuclease. A monomeric Nuclease is a Nuclease that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TALE repeats with the

catalytic domain of I-TevI described in W02012138927. TALE-nuclease have been already

described and used to stimulate gene targeting and gene modifications (Boch, Scholze et

al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010). Such engineered TAL-nucleases are commercially available under the trade name TALEN T M (Cellectis,8rue de la Croix Jarry, 75013 Paris, France).

The rare-cutting endonuclease according to the present invention can also be a Cas9 endonuclease. Recently, a new genome engineering tool has been developed based on

the RNA-guided Cas9 nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al.

2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) from the type l prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeats) adaptive immune system

(see for review (Sorek, Lawrence et al. 2013)). The CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or

plasmid. CRISPR-mediated genome engineering first proceeds by the selection of target sequence often flanked by a short sequence motif, referred as the proto-spacer adjacent motif (PAM). Following target sequence selection, a specific crRNA, complementary to this target sequence is engineered. Trans-activating crRNA (tracrRNA) required in the

CRISPR type I systems paired to the crRNA and bound to the provided Cas9 protein. Cas9 acts as a molecular anchor facilitating the base pairing of tracRNA with cRNA (Detcheva, Chylinski et al. 2011). In this ternary complex, the dual tracrRNA:crRNA structure acts as

guide RNA that directs the endonuclease Cas9 to the cognate target sequence. Target

recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence for homology between the target sequence and the crRNA. In addition to the

target sequence-crRNA complementarity, DNA targeting requires the presence of a short

motif adjacent to the protospacer (protospacer adjacent motif - PAM). Following pairing between the dual-RNA and the target sequence, Cas9 subsequently introduces a blunt

double strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

Are also encompassed in the scope of the present invention rare-cutting endonuclease variants which present a sequence with high percentage of identity or high percentage of homology with sequences of rare-cutting endonuclease described in the present

application, at nucleotidic or polypeptidic levels. By high percentage of identity or high

percentage of homology it is intended 70%, more preferably 75%, more preferably 80%,

more preferably 85%, more preferably 90%, more preferably 95, more preferably 97%, more preferably 99% or any integer comprised between 70% and 99%.

By "vector" is intended to mean a nucleic acid molecule capable of transporting another

nucleic acid to which it has been linked. A vector which can be used in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear

or circular DNA or RNA molecule which may consists of a chromosomal, non

chromosomal, semi-synthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to

which they are linked (expression vectors). Large numbers of suitable vectors are known to those skilled in the art and commercially available. Some useful vectors include, for

example without limitation, pGEM13z. pGEMT and pGEMTEasy {Promega, Madison, WI); pSTBluel (EMD Chemicals Inc. San Diego, CA); and pcDNA3.1, pCR4- TOPO, pCR-TOPO-II, pCRBlunt-II-TOPO (Invitrogen, Carlsbad, CA). Preferably said vectors are expression vectors, wherein the sequence(s) encoding the rare-cutting endonuclease of the invention is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said rare-cutting endonuclease. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said polynucleotide, a ribosome-binding site, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which the polypeptide is expressed. Preferably, when said rare-cutting endonuclease is a heterodimer, the two polynucleotides encoding each of the monomers are included in two vectors to avoid intraplasmidic recombination events. In another embodiment the two polynucleotides encoding each of the monomers are included in one vector which is able to drive the expression of both polynucleotides, simultaneously. In some embodiments, the vector for the expression of the rare-cutting endonucleases according to the invention can be operably linked to an algal-specific promoter. In some embodiments, the algal-specific promoter is an inducible promoter. In some embodiments, the algal-specific promoter is a constitutive promoter. Promoters that can be used include, for example without limitation, a Pptcal promoter (the C02 responsive promoter of the chloroplastic carbonic anyhydrase gene, ptcal, from P. tricornutum), a NITI promoter, an AMTI promoter, an

AMT2 promoter, an AMT4 promoter, a RHI promoter, a cauliflower mosaic virus 35S promoter, a tobacco mosaic virus promoter, a simian virus 40 promoter, a ubiquitin

promoter, a PBCV-1 VP54 promoter, or functional fragments thereof, or any other suitable promoter sequence known to those skilled in the art. In another more preferred

embodiment according to the present invention the vector is a shuttle vector, which can both propagate in E. coli (the construct containing an appropriate selectable marker and

origin of replication) and be compatible for propagation or integration in the genome of the selected algae.

The term "promoter" as used herein refers to a minimal nucleic acid sequence sufficient to direct transcription of a nucleic acid sequence to which it is operably linked. The term

"promoter" is also meant to encompass those promoter elements sufficient for

promoter-dependent gene expression controllable for cell-type specific expression, tissue

specific expression, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the naturally-occurring gene.

By "inducible promoter" it is mean a promoter that is transcriptionally active when bound

to a transcriptional activator, which in turn is activated under a specific conditionss, e.g.,

in the presence of a particular chemical signal or combination of chemical signals that affect binding of the transcriptional activator, e.g., CO 2 or NO 2 , to the inducible promoter

and/or affect function of the transcriptional activator itself.

The term "transfection" or "transformation" as used herein refer to a permanent or transient genetic change, preferably a permanent genetic change, induced in a cell

following incorporation of non-host nucleic acid sequences.

The term "host cell" refers to a cell that is transformed using the methods of the

invention. In general, host cell as used herein means an algal cell into which a nucleic acid target sequence has been modified.

By "catalytic domain" is intended the protein domain or module of an enzyme containing

the active site of said enzyme; by active site is intended the part of said enzyme at which

catalysis of the substrate occurs. Enzymes, but also their catalytic domains, are classified and named according to the reaction they catalyze. The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical

reactions they catalyze (http://www.chem.qmul.ac.uk/iubmb/enzyme/).

By "mutagenesis" is understood the elimination or addition of at least one given DNA

fragment (at least one nucleotide) or sequence, bordering the recognition sites of rare

cutting endonuclease.

By "NHEJ" (non-homologous end joining) is intended a pathway that repairs double

strand breaks in DNA in which the break ends are ligated directly without the need for a

homologous template. NHEJ comprises at least two different processes. Mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation

{Critchlow, 1998 #17}or via the so-called microhomology-mediated end joining (Ma, Kim et al. 2003) that results in small insertions or deletions and can be used for the creation of

specific gene knockouts.

The term "Homologous recombination" refers to the conserved DNA maintenance pathway involved in the repair of DSBs and other DNA lesions. In gene targeting

experiments, the exchange of genetic information is promoted between an endogenous

chromosomal sequence and an exogenous DNA construct. Depending of the design of the targeted construct, genes could be knocked out, knocked in, replaced, corrected or

mutated, in a rational, precise and efficient manner. The process requires homology between the targeting construct and the targeted locus. Preferably, homologous

recombination is performed using two flanking sequences having identity with the endogenous sequence in order to make more precise integration as described in

W09011354.

The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same,

this enablement being provided in particular for the subject matter of the appended

claims, which make up a part of the original description.

As used above, the phrases "selected from the group consisting of", "chosen from" and the like include mixtures of the specified materials.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all

values and sub-ranges within a numerical limit or range are specifically included as if

explicitly written out.

The above description is presented to enable a person skilled in the art to make and use

the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily

apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and

scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding can be obtained by

reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

Example 1: Increased lipid content in diatoms using TALE-Nuclease targeting the UDP glucose pyrophosphorylase (UGPase) gene

In order to determine the impact of UGPase gene inactivation on lipid content in diatoms, one engineered TALE-Nuclease to induce targeted mutagenesis in UGPase gene (SEQ ID

NO: 3) in diatoms, one engineered TALE-Nuclease, called UGP TALE-Nuclease encoded by the pCLS19745 (SEQ ID NO: 4) and pCLS19749 (SEQ ID NO: 5) plasmids designed to cleave

the DNA sequence 5'

TGCCGCCTTCGAGTCGACCTATGGTAGTCTCGTCTCGGGTGATTCCGGAA- 3' (SEQ ID NO: 6) were used. These TALE-Nuclease encoding plasmids were co-transformed with a plasmid

conferring resistance to nourseothricin (NAT) in a wild type diatom strain. The individual

clones resulting from the transformation were screened for the presence of mutagenic

events which lead to UGPase gene inactivation. The identified clones were analyzed for their lipid contents using Bodipy labeling 493/503 (Molecular Probe).

Materials and methods

Culture conditions Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown in filtered Guillard's f/2

medium without silica [(40°/° w/v Sigma Sea Salts S9883, supplemented with 1X Guillard's f/2 marine water enrichment solution (Sigma G0154)] in a Sanyo incubator (model MLR-351) at a constant temperature (20 +/- 0.5 °C). The incubator is equipped with white cold neon light tubes that produce an illumination of about 120 pmol photons

m-2 s-1 and a photoperiod of 12h light : 12h darkness (illumination period from 9AM to

9PM). Liquid cultures were made in vented cap flasks put on an orbital shaker (Polymax 1040, Heidolph) with a rotation speed of 30 revolutions min' and an angle of 5°.

Genetic transformation 5.107 cells were collected from exponentially growing liquid cultures (concentration of

about 106 cells/ ml) by centrifugation (3000 rpm for 10 minutes at 20°C). The supernatant

was discarded and the cell pellet resuspended in 500A of fresh f/2 medium. The cell suspension was then spread on the center one-third of a 10 cm 1% agar plate containing

20°/° sea salts supplemented with f/2 solution without silica. Two hours later, transformation was carried out using microparticle bombardment (Biolistic PDS-1000/He Particle Delivery System (BioRad)). The protocol is adapted from Falciatore et al., (1999)

and Apt et al., (1999) with minor modifications. Briefly, M17 tungstene particles 1.1p1m diameter, BioRad) were coated with 9pg of a total amount of DNA composed of 1.5p1g

(experiment 2) or 3pg (experiment 1) of each monomer of TALE-Nucleases (pCLS19745 and pCLS19749), 3pg of the NAT selection plasmid (pCLS16604) (SEQ ID NO: 1) and 3pg of

an empty vector (pCLS0003) (SEQ ID NO: 2) using 1.25M CaCl2 and 20mM spermidin according to the manufacturer's instructions. As a negative control, beads were coated

with a DNA mixture containing 3pg of the NAT selection plasmid (pCLS16604) and 6pg of an empty vector (pCLS0003) (SEQ ID NO: 2). Agar plates with the diatoms to be transformed were positioned at 7.5cm from the stopping screen within the bombardment

chamber (target shelf on position two). A burst pressure of 1550 psi and a vacuum of 25Hg/in were used. After bombardment, plates were incubated for 48 hours with a 12h

light: 12h dark photoperiod.

Selection Two days post transformation, bombarded cells were gently scrapped with 700pl of f/2

medium without silica and spread on two 10 cm 1% agar plates (20°/° sea salts

supplemented with f/2 medium without silica) containing 300 g ml-1 nourseothricin (Werner Bioagents). Plates were then placed in the incubator under a 12h light: 12h

darkness cycle for at least three weeks. 3 to 4 weeks after transformation, on average, resistant colonies resulting from a stable transformation were re-streaked on fresh 10 cm 1% agar plates containing 300 pg.ml-1 nourseothricin.

Characterization A-Colony screening Resistant colonies were picked and dissociated in 20Al of lysis buffer (1% TritonX-100, 20mM Tris-HCI pH8, 2mM EDTA) in an eppendorf tube. Tubes were vortexed for at least 30 sec and then kept on ice for 15 min. After heating for 10 min at 85°C, tubes were cooled down at RT and briefly centrifuged to pellet cells debris. Supernatants were used immediately or stocked at 4°C. 5Al of a 1:5 dilution in milliQ H20 of the supernatants, were used for each PCR reaction. Specific primers for TALE-Nuclease screens: TALE NucleaseFor 5'- AATCTCGCCTATTCATGGTG-3' (SEQ ID NO: 7) and HARev 5' TAATCTGGAACATCGTATGGG-3' (SEQ ID NO: 8). TALE-NucleaseFor 5' AATCTCGCCTATTCATGGTG - 3' (SEQ ID NO: 7) and STagRev 5' TGTCTCTCGAACTTGGCAGCG - 3'(SEQ ID NO: 9).

B-Identification of mutagenic events

The UGPase target was amplified using a 1:5 dilution of the colony lysates with sequence specific primers flanked by adaptators needed for HTS sequencing on a 454 sequencing system (454 Life Sciences) and the two following primers: UGP_For 5' CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag- GTTGAATCGGAATCGCTAACTCG-3' (SEQ ID NO: 10) and UGPRev 5'- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG - Tag GACTTGTTTGGCGGTCAAATCC-3'(SEQ ID NO: 11).

The PCR products were purified on magnetic beads (Agencourt AMPure XP, Beckman Coulter) and quantified with a NanoDrop 1000 spectrophotometer (Thermo Scientifioc). 50ng of the amplicons were denatured and then annealed in 10pl of the annealing buffer (10mM Tris-HCI pH8, 100mM NaCl, 1mM EDTA) using an Eppendorf MasterCycle gradient PCR machine. The annealing program is as follows: 95°C for 10 min; fast cooling to 85°C at 3°C/sec; and slow cooling to 25°C at 0.3°C/sec. The totality of the annealed DNA was digested for 15 min at 37°C with 0.5Al of the T7 Endonuclease I (10U/pl) (M0302, Biolabs) in a final volume of 20Al (1X NEB buffer 2, Biolabs). 10lA of the digestion were then loaded on a 10% polyacrylamide MiniProtean TBE precast gel (BioRad). After migration the gel was stained with SYBRgreen and scanned on a Gel Doc XR+ apparatus (BioRad).

C-Measure of the mutagenesis frequency by Deep sequencing

The UGPase target was amplified with specific primers flanked by adaptators needed for

HTS sequencing on the 454 sequencing system (454 Life Sciences) using the primer UGPFor 5'- 5'- CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag

GTTGAATCGGAATCGCTAACTCG-3'-3' (SEQ ID NO: 12) and UGPRev 5' CCTATCCCCTGTGTGCCTTGGCAGTCTCAG - GACTTGTTTGGCGGTCAAATCC -3' (SEQ ID NO:

13). 5000 to 10 000 sequences per sample were analyzed.

D- Phenotypic characterization of UDP KO clones by Bodipy labeling

Cells were re-suspended at the density of 5.10s cells/ml and washed twice in culture

medium (filtered Guillard's f/2 medium without silica). The bodipy labeling was performed with 10pM of final concentration of Bodipy 493/503 (Molecular Probe) in presence of 10% of DMSO during 10 minutes at room temperature in the dark. The

fluorescence intensity was measured by flow cytometry at 488nM (MACSQuant Analyzer,

Miltenyi Biotec).

E- Lipid content analysis

The lipid content analysis was performed by the APLILIPID company (Applied Lipidomics Investigation) using protocol previously described in (Vieler, Wilhelm et al. 2007;

Lamaziere, Wolf et al. 2012; Lamaziere, Wolf et al. 2013).

Results

Three independent experiments were performed using the TALE-Nuclease targeting the

UGPase gene. For each of them, the presence of mutagenic events in the clones obtained three weeks after diatoms transformation was analyzed.

For the first experiment, 18 clones were obtained in the condition corresponding to diatoms transformed with TALE-Nuclease encoding plasmids (condition 1). Finally, 6

clones resulting from the transformation with the empty vector were obtained (condition 2). The UGPase target amplification was performed on 12 clones obtained in the condition 1 and 2 clones obtained in the condition 2. On the 12 clones tested, 4 present a

PCR band higher than expected showing a clear mutagenic event, 1 presents no

amplification of the UGPase target, 7 present a band at the wild type size. A T7 assay was assessed on these 12 clones (Figure 4). One clone among them was positive in T7 assay

which reflects the presence of mutagenic events (Figure 5). As expected no signal was

detected in the 2 clones from the condition corresponding to empty vector (condition 2).

For the second experiment, 62 clones were obtained in the condition corresponding to diatoms transformed with TALE-Nuclease encoding plasmids (condition 1). Among them,

36 were tested for the presence of the DNA sequences encoding both TALE-Nuclease monomers. 11/36 (i.e. 30.5%) were positive for both TALE-Nuclease monomers DNA sequences. Finally, 38 clones resulting from the transformation with the empty vector

were obtained (condition 2). The UGPase target amplification was performed on 11

clones obtained in the condition 1 and 2 clones obtained in the condition 2. On the 11

clones tested, 5 present no amplification of the UGPase target, 6 present a band at the wild type size (Figure 6).

In order to identify the nature of the mutagenic event in the 4 clones displaying a higher

PCR amplification product from experiment 1 (Figure 4), we sequenced these fragments. All of them present an insertion of 261bp (37-5A3), 228bp (37-7A1), 55bp (37-7B2) and

330bp (37-16A1), respectively leading to the presence of stop codon in the coding

sequence. The clone 37-3B4 presenting a positive signal for T7 assay was characterized by Deep sequencing. The mutagenesis frequency in this clone was 86% with several type of

mutagenic event (either insertion or deletion). An example of mutated sequences is presented in Figure 7.

To investigate the impact of UGPase gene inactivation on lipid content, a Bodipy labeling

(Molecular Probe) was performed on one clone harboring a mutagenic event in the

UGPase target (37-7A). In parallel, the Phaeodactylum tricornutum wild type strain and one clone resulting from the transformation with the empty vector were tested. The

results are presented in Figure 8. We observed an increase of the fluorescence intensity in the clone presenting an inactivation of the UGPase gene compared to the two control strains. This experiment was reproduced 3 times and a shift in the fluorescence intensity

was observed at each time. As Bodipy labeling reflects the lipid content of the cells, these

results demonstrated a robust and reproducible increase of the lipid content of the mutated strains.

In order to perform quantitative analysis of the fatty acid (FA) and the triacylglycerol (TAG) content, the wet pellets of diatoms corresponding to the mutants 37-7A1 and its

associated controls empty vector and Phaeodactylum tricornutum wild type were brought to the APlipid company for an extensive lipidomic analysis. When compared to the

controls (Pt-wt parental strain and resistant clone), the mutant 37-7A1 (UGPase) presents a 2 fold increase of its FA content reported to the total number of cells. The content of TAG (in nmoles and reported to the total number of cells) is increased by a factor 24 for

the mutant 37-7A1 (Figure 9).

Thus, a TALE nuclease targeting the UGPase gene induces a reproducible (2 independent experiments), and at high frequency, targeted mutagenesis (up to 100%). Moreover, the

inactivation of the UGPase gene leads to a strong and reproducible increase of lipid content in bodipy labeling. The quantification reveals an increase of 2 fold of fatty acid

and 24 fold of TAG in the clone UGPase knock out compare to controls.

Example 2: Targeted mutagenesis induced by a TALE-Nuclease targeting a putative

elongase gene

In order to determine the impact of the putative elongase gene (SEQ ID NO: 14)

inactivation on lipid content in diatoms, one engineered TALE-Nuclease, called elongaseTALE-Nuclease encoded by the pCLS19746 (SEQ ID NO: 15) and pCLS19750 (SEQ

ID NO: 16) plasmids designed to cleave the DNA sequence 5' -

TCTTTTCCCTCGTCGGCatgctccggacctttCCCCAGCTTGTACACAA - 3' (SEQ ID NO: 17) was

used. Although this TALE-nuclease targets a sequence coding a protein with unknown

function, this target presents 86% of sequence identity with the mRNA of the fatty acid elongase 6 (ELOVL6) in Taeniopygia guttata, and 86% of sequence identity with the elongation of very long chain fatty acids protein 6-like (LOC100542840) in meleagris

gallopavo.

These TALE-Nuclease encoding plasmids were co-transformed with a plasmid conferring resistance to nourseothricin (NAT) in a wild type diatom strain. The individual clones

resulting from the transformation were screened for the presence of mutagenic events which lead to elongase gene inactivation.

Materials and methods

Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown and transformed according to the methods described in example 1 with M17 tungstene particles (1.1p1m

diameter, BioRad) coated with 9pg of a total amount of DNA composed of 1.51g of each monomer of TALE-Nucleases (pCLS19746 (SEQID NO: 15) and pCLS19750 (SEQID NO: 16),

3pg of the NAT selection plasmid (pCLS16604) (SEQ ID NO: 1) and 3pg of an empty vector (pCLS0003) (SEQ ID NO: 2) using 1.25M CaCl2 and 20mM spermidin according to the

manufacturer's instructions.

Characterization

A-Colony screening

After selection, resistant colonies were picked and dissociated according to the method described in example 1. Supernatants were used were used for each PCR reaction.

Specific primers for TALE-Nuclease screens: TALE-NucleaseFor 5' AATCTCGCCTATTCATGGTG-3' (SEQ ID NO: 7) and HARev 5' TAATCTGGAACATCGTATGGG-3' (SEQ ID NO: 8). TALE-NucleaseFor 5' AATCTCGCCTATTCATGGTG - 3' (SEQ ID NO: 7) and S-TagRev 5'

TGTCTCTCGAACTTGGCAGCG - 3'(SEQ ID NO: 9).

B-Identification of mutagenic event

The elongase target was amplified using a 1:5 dilution of the lysis colony with sequence specific primers flanked by adaptators needed for HTS sequencing on the 454 sequencing

system (454 Life Sciences) and the two following primers: elongaseFor 5' CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag-AAGCGCATCCGTTGGTTCC-3'(SEQ ID NO: 18)

and elongaseRev 5'- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG

TCAATGAGTTCACTGGAAAGGG -3'(SEQ ID NO: 19).

The PCR products were purified on magnetic beads (Agencourt AMPure XP, Beckman Coulter) and quantified with a NanoDrop 1000 spectrophotometer (Thermo Scientifioc).

50ng of the amplicons were denatured and then annealed in 10pl of annealing buffer (10mM Tris-HCI pH8, 100mM NaCl, 1mM EDTA) using an Eppendorf MasterCycle gradient

PCR machine. The annealing program is as follows: 95°C for 10 min; fast cooling to 85°C at

3°C/sec; and slow cooling to 25°C at 0.3°C/sec. The totality of the annealed DNA was digested for 15 min at 37°C with 0.5Al of the T7 Endonuclease I (10U/pl) (M0302 Biolabs) in a final volume of 20Al (1X NEB buffer 2, Biolabs). 10lA of the digestion were then

loaded on a 10% polyacrylamide MiniProtean TBE precast gel (BioRad). After migration

the gel was stained with SYBRgreen and scanned on a Gel Doc XR+ apparatus (BioRad).

C-Measure of the mutagenesis frequency by Deep sequencing

The elongase target was amplified with sequence specific primers flanked by adaptators needed for HTS sequencing on the 454 sequencing system (454 Life Sciences) using the

primer Delta 6 elongase_For 5'- AAGCGCATCCGTTGGTTCC -3' (SEQID NO: 20) and Delta 6

elongaseRev 5'- TCAATGAGTTCACTGGAAAGGG -3' (SEQ ID NO: 21). 5000 to 10 000 sequences per sample were analyzed.

D- Lipid content analysis

The lipid content analysis was performed by the APLILIPID company (Applied Lipidomics

Investigation) using protocol previously described in (Vieler, Wilhelm et al. 2007;

Lamaziere, Wolf et al. 2012; Lamaziere, Wolf et al. 2013).

Results

Three weeks after the transformation of the diatoms, 62 clones were obtained in the condition corresponding to the transformation performed with the TALE-Nuclease

encoding plasmids (condition 1). Among them, 35 were tested for the presence of both TALE-Nuclease monomers DNA sequences. 11/27 (i.e. 40.7%) were positive for both TALE

Nuclease monomers DNA sequences. Finally, 38 clones resulting from the transformation

with the empty vector were obtained (condition 2).

The 11 clones, positive for both TALE-Nuclease monomers DNA sequences were tested with the T7 assay. The Phaeodactylum tricornutum wild type strain, as well as four clones

resulting from the transformation with the empty vector, were tested in parallel. Four clones presented no amplification. Because the amplification of another locus is possible,

the quality of the lysates is not questioned. So the absence of amplification could suggest

the presence of a large mutagenic event at the elongase locus. One clone showed in equal proportions a PCR product at the expected size and another one with a higher weight, actually demonstrating a clear mutagenic event (Figure 10). One clone was positive in the

T7 assay, which reflects the presence of mutagenic events and 9 clones presented no

signal in the T7 assay. As expected no signal was detected in the condition corresponding to the empty vector or the Phaeodactylum tricornutum wild type strain.

In order to identify the nature of the mutagenic event in the clone displaying a higher PCR amplification product, we sequenced this fragment. An insertion of 83 bp was detected

leading to presence of stop codon in the coding sequence. The clone presenting a positive T7 signal was characterized by Deep sequencing. The mutagenesis frequency in this clone

was 5.9% with one type of mutation (deletion of 22bp). An example of mutated sequences is presented in Figure 11.

In order to perform quantitative analysis of the fatty acid (FA) and the triacylglycerol

(TAG) content, the wet pellets of diatoms corresponding to the mutant and its associated

control empty vector, were brought to the APlipid company for an extensive lipidomic analysis. When compared to the control (resistant clone), the mutant (Elongase) presents

a 3 fold increase of its FA content reported to the total number of cells. The content of

TAG (in nmoles and reported to the total number of cells) is increased by a factor 3 for the mutant elongase (Figure 12).

Thus, a TALE nuclease targeting the Elongase gene induces a high frequency of targeted

mutagenesis (up to 50%). To investigate the impact of Elongase gene inactivation on lipid profile, the sub-cloning of the clone with 50% of mutated event will be done. The

quantification of lipid content in this clone reveals an increase of 3 fold of fatty acid and 3

fold of TAG in the clone Elongase mutant compare to control.

Example 3: Targeted mutagenesis induced by a TALE-Nuclease targeting the G3PDH

gene

In order to determine the impact of the Glycerol-3 Phosphatedeshydrogenase (G3PDH) gene (SEQ ID NO: 22) inactivation on lipid content in diatoms, one engineered TALE

Nuclease, called G3PDHTALE-Nuclease encoded by the pCLS23159 (SEQ ID NO: 23) and pCLS23163 (SEQ ID NO: 24) plasmids designed to cleave the DNA sequence 5' TTCTGACCAACTCGATAAAGTATGCATCATCGGTAGCGGTAACTGGGGAA - 3' (SEQ ID NO: 25) was used. These TALE-Nuclease encoding plasmids were co-transformed with a plasmid

conferring resistance to nourseothricin (NAT) in a wild type diatom strain. The individual

clones resulting from the transformation were screened for the presence of mutagenic events which lead to G3PDH gene inactivation.

Materials and methods

Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown and transformed according to the methods described in example 1 with M17 tungstene particles (1.1p1m

diameter, BioRad) coated with 9pg of a total amount of DNA composed of 3pg of each monomer of TALE-Nucleases (pCLS23159 (SEQ ID NO: 23) and pCLS23163 (SEQ ID NO: 24)), 3pg of the NAT selection plasmid (pCLS16604) (SEQ ID NO: 1) and 3pg of an empty

vector (pCLS0003) (SEQ ID NO: 2) using 1.25M CaCl2 and 20mM spermidin according to

the manufacturer's instructions. As negative control, beads were coated with a DNA mixture containing 3pg of the NAT selection plasmid (pCLS16604) and 6pg of an empty vector (pCLS0003) (SEQ ID NO: 2). Agar plates with the diatoms to be transformed were positioned at 7.5cm from the stopping screen within the bombardment chamber (target shelf on position two). A burst pressure of 1550 psi and a vacuum of 25Hg/in were used. After bombardment, plates were incubated for 48 hours with a 12h light: 12h dark photoperiod.

Characterization

A-Colony screening

After selection, resistant colonies were picked and dissociated according to the methods

described in example 1. Supernatants were used for each PCR reaction. Specific primers

for TALE-Nuclease screens: TALE-Nuclease_For 5'- AATCTCGCCTATTCATGGTG-3' (SEQ ID NO: 7) and Stag_Rev 5'- TGTCTCTCGAACTTGGCAGCG - 3' (SEQ ID NO: 9). HAFor 5'

ACCCATACGATGTTCCAGATTACGCT - 3' (SEQ ID NO: 26) and TALE-NucleaseRev 5' AATCTTGAGAAGTTGGCCTGTGTC - 3'(SEQ ID NO: 27).

B-Identification of mutagenic event by Deep sequencing

The G3PDH target was amplified using a 1:5 dilution of the lysis colony with sequence

specific primers flanked by adaptators needed for HTS sequencing on the 454 sequencing

system (454 Life Sciences) and the two following primers: G3PDH_For 5' CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag- TCTGCTACTGCTCATCCGCACC -3' (SEQ ID

NO: 28) and G3PDHRev 5'- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG TCGCGACAGGCTTCTGCTAGATC-3' (SEQ ID NO: 29). 5000 to 10 000 sequences per sample

were analyzed.

E- Lipid content analysis

The lipid content analysis was performed by the APLILIPID company (Applied Lipidomics

Investigation) using protocol previously described in (Vieler, Wilhelm et al. 2007; Lamaziere, Wolf et al. 2012; Lamaziere, Wolf et al. 2013).

Results

Three weeks after the transformation of the diatoms, 13 clones were obtained in the condition corresponding to the transformation performed with the TALE-Nuclease

encoding plasmids (condition 1). Among them, 7 were tested for the presence of both TALE-Nuclease monomers DNA sequences. 7/13 (i.e. 53.8%) were positive for both TALE Nuclease monomers DNA sequences. Among them, one present 33% of frequency of

targeted mutagenesis at the recognition TALE-Nuclease site. An example of mutated

sequences is presented in Figure 13. As expected no signal was detected in the condition corresponding to the empty vector or the Phaeodactylum tricornutum wild type strain.

Thus, a TALE nuclease targeting the G3PDH gene induces a high frequency of targeted mutagenesis (up to 33%).

Example 4: Targeted mutagenesis induced by a TALE-Nuclease targeting the Omega3 desaturase gene

In order to determine the impact of the Omega 3desaturase gene (SEQ ID NO: 30)

inactivation on lipid content in diatoms, one engineered TALE-Nuclease, called Omega3 desaturaseTALE-Nuclease encoded by the pCLS23158 (SEQ ID NO: 31) and pCLS23162

(SEQ ID NO: 32) plasmids designed to cleave the DNA sequence 5'

TTTTCCACAACACTGTTAATGCCTTTTCGTTGCGCATACCGAGTACCCA- 3' (SEQ ID NO: 33) was used. These TALE-Nuclease encoding plasmids were co-transformed with a plasmid conferring resistance to nourseothricin (NAT) in a wild type diatom strain. The individual

clones resulting from the transformation were screened for the presence of mutagenic

events which lead to Omega3 desaturase gene inactivation.

Materials and methods

Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown and transformed

according to the method described in example 1, with M17 tungstene particles (1.1p1m diameter, BioRad) coated with 9pg of a total amount of DNA composed of 1.51g of each

monomer of TALE-Nucleases (pCLS23158 (SEQ ID NO: 31) and pCLS23162 (SEQ ID NO: 32)), 3pg of the NAT selection plasmid (pCLS16604) (SEQ ID NO: 1) and 3pg of an empty vector (pCLS0003) (SEQ ID NO: 2) using 1.25M CaCl2 and 20mM spermidin according to the manufacturer's instructions. As negative control, beads were coated with a DNA mixture containing 3pg of the NAT selection plasmid (pCLS16604) and 6pg of an empty vector (pCLS0003) (SEQ ID NO: 2).

Characterization

A-Colony screening

After selection, resistant colonies were picked and dissociated according to the method described in example 1. Supernatants were used for each PCR reaction. Specific primers

for TALE-Nuclease screens: TALE-NucleaseFor 5'- AATCTCGCCTATTCATGGTG-3' (SEQ ID

NO: 7) and StagRev 5'- TGTCTCTCGAACTTGGCAGCG - 3' (SEQ ID NO: 9). HAFor 5' ACCCATACGATGTTCCAGATTACGCT - 3' (SEQ ID NO: 26) and TALE-NucleaseRev 5'

AATCTTGAGAAGTTGGCCTGTGTC - 3'(SEQ ID NO: 27).

B-Identification of mutagenic event by Deep sequencing

The Omega3 desaturase target was amplified using a 1:5 dilution of the lysis colony with sequence specific primers flanked by adaptators needed for HTS sequencing on the 454

sequencing system (454 Life Sciences) and the two following primers: Omega3

desaturaseFor 5'- CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag GCGTGTGCTCACCTGTTGTCC -3' (SEQ ID NO: 34) and Omega3 desaturase _Rev 5'

CCTATCCCCTGTGTGCCTTGGCAGTCTCAG - AAGCATGCGCTTCACTTCGCTC -3' (SEQ ID NO: 35). 5000 to 10 000 sequences per sample were analyzed.

Results

Three weeks after the transformation of the diatoms, 9 clones were obtained in the condition corresponding to the transformation performed with the TALE-Nuclease

encoding plasmids (condition 1). Among them, 6 were tested for the presence of both TALE-Nuclease monomers DNA sequences. 6/9 (i.e. 66%) were positive for both TALE

Nuclease monomers DNA sequences. The targeted mutagenesis frequency was

determined by Deep sequencing on 3 out of the 6 clones. All of them present a high frequency of mutagenic event at the TALE-Nuclease recognition site: 14; 70 and 90%. An example of mutated sequences is presented in Figure 14. As expected no signal was detected in the condition corresponding to the empty vector or the Phaeodactylum tricornutum wild type strain.

Thus, a TALE nuclease targeting the Omega3 desaturase gene induces a high frequency of

targeted mutagenesis (up to 90%).

Example 5: Targeted mutagenesis induced by a TALE-Nuclease targeting the putative

palmitoyl protein thioesterase gene (PPT)

In order to determine the impact of the PPT gene (SEQ ID NO: 36) inactivation on lipid

content in diatoms, one engineered TALE-Nuclease, called PPTTALE-Nuclease encoded by the pCLS19744 (SEQ ID NO: 37) and pCLS19748 (SEQ ID NO: 38) plasmids designed to

cleave the DNA sequence 5'

TGGTCTTTGCCCCATGGGATGGGAGACGTGCTAACTGGATGCAA- 3' (SEQ ID NO: 39) was used. These TALE-Nuclease encoding plasmids were co-transformed with a plasmid

conferring resistance to nourseothricin (NAT) in a wild type diatom strain. The individual

clones resulting from the transformation were screened for the presence of mutagenic

events which lead to PPT gene inactivation.

Materials and methods

Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown and transformed

according to the method described in example 1, with M17 tungstene particles (1.1p1m diameter, BioRad) coated with 9pg of a total amount of DNA composed of 1.51g of each

monomer of TALE-Nucleases (pCLS19744 (SEQ ID NO: 37) and pCLS19748 (SEQ ID NO: 38)), 3pg of the NAT selection plasmid (pCLS16604) (SEQ ID NO: 1) and 3pg of an empty vector (pCLS0003) (SEQ ID NO: 2) using 1.25M CaCl2 and 20mM spermidin according to

the manufacturer's instructions. As negative control, beads were coated with a DNA

mixture containing 3pg of the NAT selection plasmid (pCLS16604) and 6pg of an empty vector (pCLS0003) (SEQ ID NO: 2).

Characterization

A-Colony screening

After selection, resistant colonies were picked and dissociated according to the method

described in example 1. Supernatants were used for each PCR reaction. Specific primers for TALE-Nuclease screens: TALE-NucleaseFor 5'- AATCTCGCCTATTCATGGTG-3' (SEQ ID

NO: 7) and Stag_Rev 5'- TGTCTCTCGAACTTGGCAGCG - 3' (SEQ ID NO: 9). HAFor 5' ACCCATACGATGTTCCAGATTACGCT - 3' (SEQ ID NO: 26) and TALE-NucleaseRev 5'

AATCTTGAGAAGTTGGCCTGTGTC - 3'(SEQ ID NO: 27).

B-Identification of mutagenic event by Deep sequencing

The PPT target was amplified using a 1:5 dilution of the lysis colony with sequence specific

primers flanked by adaptators needed for HTS sequencing on the 454 sequencing system (454 Life Sciences) and the two following primers: PPTFor 5'

CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag-GAAGAACAGTCGCACCTGGTGC -3' (SEQ ID NO: 40) and PPTRev 5'- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG TCCGCCCTAACACCTTCCGC -3' (SEQ ID NO: 41). 5000 to 10 000 sequences per sample

were analyzed.

Results

Three weeks after the transformation of the diatoms, 11 clones were obtained in the condition corresponding to the transformation performed with the TALE-Nuclease encoding plasmids (condition 1). Among them 3/11 (i.e. 27.3%) were positive for both

TALE-Nuclease monomers DNA sequences. The targeted mutagenesis frequency was determined by Deep sequencing on 1 out of the 3 clones. This clone presents a high

frequency of mutagenic event at the TALE-Nuclease recognition site: 22%. An example of mutated sequences is presented in Figure 15. As expected no signal was detected in the

condition corresponding to the empty vector or the Phaeodactylum tricornutum wild type strain.

Thus, a TALE nuclease targeting the PPT gene induces a high frequency of targeted mutagenesis (up to 22%).

Example 6: Targeted mutagenesis induced by a TALE-Nuclease targeting the Enoyl ACP reductase gene

In order to determine the impact of the Enoyl ACP reductase gene (SEQ ID NO: 42)

inactivation on lipid content in diatoms, one engineered TALE-Nuclease, called EnoylACPReductaseTALE-Nuclease encoded by the pCLS23157 (SEQ ID NO: 43) and

pCLS23161 (SEQ ID NO: 44) plasmids designed to cleave the DNA sequence 5'

TGTTGCCGATTCCACTGGTTACGGCTGGGCGATCGCCAAAGCTTTGGCCGAAGCAGGA - 3' (SEQ ID NO: 45) was used. These TALE-Nuclease encoding plasmids were co-transformed with a

plasmid conferring resistance to nourseothricin (NAT) in a wild type diatom strain. The individual clones resulting from the transformation were screened for the presence of

mutagenic events which lead to Enoyl ACP reductase gene inactivation.

Materials and methods

Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown and transformed

according to the method described in example 1, with M17 tungstene particles (1.1p1m diameter, BioRad) coated with 9pg of a total amount of DNA composed of 1.51g of each

monomer of TALE-Nucleases (pCLS23157 (SEQ ID NO: 43) and pCLS23161 (SEQ ID NO: 44), 3pg of the NAT selection plasmid (pCLS16604) (SEQ ID NO: 1) and 3pg of an empty vector

(pCLS0003) (SEQ ID NO: 2) using 1.25M CaCl2 and 20mM spermidin according to the manufacturer's instructions. As negative control, beads were coated with a DNA mixture

containing 3pg of the NAT selection plasmid (pCLS16604) and 6pg of an empty vector

(pCLS0003) (SEQ ID NO: 2).

Characterization

A-Colony screening

After selection, resistant colonies were picked and dissociated according to the method described in example 1. Supernatants were used for each PCR reaction. Specific primers

for TALE-Nuclease screens: TALE-NucleaseFor 5'- AATCTCGCCTATTCATGGTG-3' (SEQ ID NO: 7) and Stag_Rev 5'- TGTCTCTCGAACTTGGCAGCG - 3' (SEQ ID NO: 9). HAFor 5' ACCCATACGATGTTCCAGATTACGCT - 3' (SEQ ID NO: 26) and TALE-NucleaseRev 5' AATCTTGAGAAGTTGGCCTGTGTC - 3'(SEQ ID NO: 27).

B-Identification of mutagenic event by Deep sequencing

The Enoyl ACP reductase target was amplified using a 1:5 dilution of the lysis colony with sequence specific primers flanked by adaptators needed for HTS sequencing on the 454

sequencing system (454 Life Sciences) and the two following primers: Enoyl ACP reductase _For 5'- CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag

GGACTGTTTCGCTACGGTACATC -3' (SEQ ID NO: 46) and Enoyl ACP reductase _Rev 5'

CCTATCCCCTGTGTGCCTTGGCAGTCTCAG - GAAATGGTGTATCCGTCCAATCC -3' (SEQ ID NO: 47). 5000 to 10 000 sequences per sample were analyzed.

Results

Three weeks after the transformation of the diatoms, 14 clones were obtained in the

condition corresponding to the transformation performed with the TALE-Nuclease

encoding plasmids (condition 1). Among them 2/14 (i.e. 14%) were positive for both TALE Nuclease monomers DNA sequences. The targeted mutagenesis frequency was determined by Deep sequencing on 1 out of the 2 clones. This clone presents a frequency

of mutagenic event at the TALE-Nuclease recognition site: 12%. An example of mutated

sequences is presented in Figure 16. As expected no signal was detected in the condition corresponding to the empty vector or the Phaeodactylum tricornutum wild type strain.

Thus, a TALE nuclease targeting the Enoyl ACP reductase gene induces a high frequency of targeted mutagenesis (up to 12%).

Example 7: Targeted mutagenesis induced by a TALE-Nuclease targeting the Delta 12 fatty acid desaturase gene

In order to determine the impact of the Delta 12 fatty aciddesaturase gene (SEQ ID NO:

48) inactivation on lipid content in diatoms, one engineered TALE-Nuclease, called Delta 12 desaturaseTALE-Nuclease encoded by the pCLS19743 (SEQID NO: 49) and pCLS19747

(SEQ ID NO: 50) plasmids designed to cleave the DNA sequence 5'

TAGCTCCCAAGAGTGCCACCAGCTCTACTGGCAGTGCTACCCTTAGCCAA- 3' (SEQ ID NO: 51) was used. These TALE-Nuclease encoding plasmids were co-transformed with a plasmid

conferring resistance to nourseothricin (NAT) in a wild type diatom strain. The individual clones resulting from the transformation were screened for the presence of mutagenic

events which lead to Delta 12 fatty acid desaturase gene inactivation.

Materials and methods

Phaeodactylum tricornutum Bohlin clone CCMP2561 was grown and transformed

according to the method described in example 1 with M17 tungstene particles (1.1pm diameter, BioRad) coated with 9pg of a total amount of DNA composed of 1.51g of each

monomer of TALE-Nucleases (pCLS19743 (SEQ ID NO: 49) and pCLS19747 (SEQ ID NO: 50)), 3pg of the NAT selection plasmid (pCLS16604) (SEQ ID NO: 1) and 3pg of an empty

vector (pCLS0003) (SEQ ID NO: 2) using 1.25M CaCl2 and 20mM spermidin according to the manufacturer's instructions. As negative control, beads were coated with a DNA

mixture containing 3pg of the NAT selection plasmid (pCLS16604) and 6pg of an empty

vector (pCLS0003) (SEQ ID NO: 2).

Characterization

A-Colony screening

After selection, resistant colonies were picked and dissociated according to the method described in example 1. Supernatants were used for each PCR reaction. Specific primers

for TALE-Nuclease screens: TALE-NucleaseFor 5'- AATCTCGCCTATTCATGGTG-3' (SEQ ID

NO: 7) and Stag_Rev 5'- TGTCTCTCGAACTTGGCAGCG - 3' (SEQ ID NO: 9). HAFor 5'-

ACCCATACGATGTTCCAGATTACGCT - 3' (SEQ ID NO: 26) and TALE-NucleaseRev 5' AATCTTGAGAAGTTGGCCTGTGTC - 3'(SEQ ID NO: 27).

B-Identification of mutagenic event by Deep sequencing

The Delta 12 fatty acid desaturase target was amplified using a 1:5 dilution of the lysis colony with sequence specific primers flanked by adaptators needed for HTS sequencing

on the 454 sequencing system (454 Life Sciences) and the two following primers: Delta12 desaturaseFor 5'- CCATCTCATCCCTGCGTGTCTCCGACTCAG-Tag

CTCGTCGGTGGTCCGTATTGG -3' (SEQ ID NO: 52) and Delta12 desaturase _Rev 5' CCTATCCCCTGTGTGCCTTGGCAGTCTCAG - TGGCGAGATCGCGCATCAGG -3' (SEQ ID NO:

53). 5000 to 10 000 sequences per sample were analyzed.

Results

Three weeks after the transformation of the diatoms, the clones obtained corresponding

to the transformation performed with the TALE-Nuclease encoding plasmids (condition 1) were screened for the presence of both TALE-Nuclease monomers DNA sequences. The

targeted mutagenesis frequency would be determined by Deep sequencing on the positive clones.

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eolf-seql.txt SEQUENCE LISTING <110> Cellectis

<120> MODIFIED DIATOMS FOR BIOFUEL PRODUCTION <130> P81307273PCT00

<150> PA201370354 <151> 2013-06-25

<160> 53 <170> PatentIn version 3.5

<210> 1 <211> 4246 <212> DNA <213> artificial sequence

<220> <223> pCLS16604

<400> 1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagctcga aattaaccct cactaaaggg aacaaaagct ggtaccccgc tttggtttca 2220 cagtcaggaa taacactagc tcgtcttcac catggatgcc aatctcgccc attcatggtg 2280 tataaaagtt caacatccaa agctagaact tttggaaaga gaaagaatgt ccgaataggg 2340 cacggcgtgc cgtattgttg gagtggacta gcagaaagtg aggaaggcac aggatgagtt 2400 tcctcgagac acatagcttc agcgtcgtgt aggctaggca gaggtgagtt ttctcgagac 2460 ataccttcag cgtcgtcttc actgtcacag tcaactgaca gtaatcgttg atccggagag 2520 Page 1 eolf-seql.txt attcaaaatt caatctgttt ggacctggat aagacacaag agcgacatcc tgacatgaac 2580 gccgtaaaca gcaaatcctg gttgaacacg tatccttttg ggggcctcca gctacgacgc 2640 tcgccccagc tggggcttcc ttactataca cagcgcatat ttcacggttg ccagaagtca 2700 agtcgaggtc gatccatatg accactcttg acgacacggc ttaccggtac cgcaccagtg 2760 tcccggggga cgccgaggcc atcgaggcac tggatgggtc cttcaccacc gacaccgtct 2820 tccgcgtcac cgccaccggg gacggcttca ccctgcggga ggtgccggtg gacccgcccc 2880 tgaccaaggt gttccccgac gacgaatcgg acgacgaatc ggacgacggg gaggacggcg 2940 acccggactc ccggacgttc gtcgcgtacg gggacgacgg cgacctggcg ggcttcgtgg 3000 tcgtctcgta ctccggctgg aaccgccggc tgaccgtcga ggacatcgag gtcgccccgg 3060 agcaccgggg gcacggggtc gggcgcgcgt tgatggggct cgcgacggag ttcgcccgcg 3120 agcggggcgc cgggcacctc tggctggagg tcaccaacgt caacgcaccg gcgatccacg 3180 cgtaccggcg gatggggttc accctctgcg gcctggacac cgccctgtac gacggcaccg 3240 cctcggacgg cgagcaggcg ctctacatga gcatgccctg cccctgagcg gccgacggta 3300 tcgataagct tgatatcgaa ttcctgcagc ccgggggatc cactagttct agagcggccg 3360 caacaactac ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg 3420 tgctatcgaa ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca 3480 cgaacagttg ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact 3540 ccgctttggt ttcacagtca ggaataacac tagctcgtct tcaccgcggt ggagctccaa 3600 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg 3660 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 3720 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 3780 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3840 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3900 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 3960 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 4020 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4080 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 4140 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 4200 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttag 4246

<210> 2

<211> 5428 <212> DNA <213> artificial sequence <220> <223> pCLS003 <400> 2 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 960 agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca 1020 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 1080 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 1140 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 1200 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 1260 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 1320 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 1380 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 1440 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 1500 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 1560 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 1620 Page 2 eolf-seql.txt acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 1680 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 1740 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 1800 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 1860 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 1920 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 1980 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 2040 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 2100 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 2160 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 2220 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 2280 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 2340 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 2400 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 2460 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 2520 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 2580 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 2640 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 2700 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 2760 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 2820 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 2880 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 2940 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac 3000 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 3060 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 3120 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 3180 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 3240 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 3300 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 3360 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 3420 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3480 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 3540 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 3600 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3660 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3720 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3780 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3840 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 3900 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 3960 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 4020 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 4080 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 4140 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 4200 cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 4260 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 4320 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 4380 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 4440 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 4500 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 4560 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 4620 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 4680 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 4740 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 4800 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 4860 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 4920 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 4980 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 5040 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 5100 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 5160 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 5220 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 5280 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 5340 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 5400 atttccccga aaagtgccac ctgacgtc 5428

<210> 3

Page 3 eolf-seql.txt <211> 3613 <212> DNA <213> Phaeodactylum tricornutum <220> <223> UDP-Glucose-Pyrophosphorylase/Phosphoglucomutase (UGP/PGM) (PHATRDRAFT_5044) <400> 3 ttcgaaagac gaacaacgag cctcccgaat atcctacggt tggttgctta ataacattgc 60 tttgacaact caagatgcct tctttcgatc ccattcgtgc aaaaatggaa gccggaggct 120 gtgctccatc ggcgattgcc gccttcgagt cgacctatgg tagtctcgtc tcgggtgatt 180 ccggaatgat tttggaagac tctattgcgc ccgtccccca gctggacaag accgcggagc 240 tggatattgc acccaacgcc acccttcttg ccgagacggt agttctcaaa ctcaatggtg 300 gactcggcac gggtatgggt ctggacaagg ccaagtccct gttgccagtc aagggggacg 360 acaccttttt ggatttgacc gccaaacaag tcattcaaat gcgtaaggaa tacggtttga 420 acgtcaagtt tatgctcatg aattcgtttt ctacttccga cgataccttg agctttttga 480 gttccaaata ccctgatctt gcttccgagg aaggtttaga aatgatgcaa aataaggtcc 540 ccaagttgaa cgcggagact ctcgagccgg catcttgtga atccgatccg gaaaatgagt 600 ggtgtccgcc gggacacggt gacttgtacg cggccttggt tggctctggt cgtcttgatg 660 ccctgctcaa ggaagggttc aaatatatgt ttgtctccaa ttcggacaac cttggtgcta 720 gcctggacct tgaaattctg acttactttg ccgagaagaa tgtacccttc ttgatggagt 780 gctgcgaacg tacagaaaac gacaaaaagg gagggcactt ggccgtccgc aaatccgatg 840 gacaacttat tcttcgggaa tctgctatgt gcgctgaaga ggatgaagat gcattcagtg 900 atatcagcaa gcaccgcttt ttcaacacca acaatttgtg ggttcgtctc gataaactca 960 aggagatcat cgaccgcaat ggcggcttta ttcctctgcc catgatcaaa aacaaaaaga 1020 cggtcgaccc caaggacgac tcgtcgaccc cggtactgca gttggaaacc gctatgggtg 1080 ccgctattga atgtttcgaa ggcgccagcg cggtggttgt tcctcgcaca cgctttgcgc 1140 ccgtcaaaaa gtgcagcgat ctgctcttgc tgcgctccga tgcatacttg ctcgtggacc 1200 acaagccggt actcaatcca gcctgcaacg ggagcgcgcc cgtgatcaat ctcgacagca 1260 aactatacaa gctggtcggc gccttggaag aagcaaccca ggacggcatt ccgtccctcg 1320 tcaagtgcga caaattgact atcaagggtt tggtccggat gtcgaaaaag accaagtttg 1380 tgggtgatgt caagattgtc aactcgagcg ccgaatctaa gtttgtgccc accggtgaag 1440 taacagggga acacgatctg acgtctaatg ctggtcttgg caagctaaag cccacctctg 1500 tttcaacagc accaattgcg ggacaaaagc ctggtacttc aggactccgg aagaaggttg 1560 ccgaattcaa gaaggaaaac taccttaaca attttgtaca agctgctttt gacgccatca 1620 aggccagtgg tacggacata tcgaaggggt ccttggtaat tggtggtgat ggtcgctact 1680 tcaaccctga agcaatccaa atacttattc agatgggtgt tgctaacggc gtcagacgtt 1740 tctggattgg acaggacggc ctcttgtcga cacccgccgt ttctgcgatc attcgggaag 1800 gcggcccgcg ttggcaaaag gcatttggag cctttatttt gacggctagt cacaatcccg 1860 gtggcccaac ggaagatttt ggtatcaagt acaactgcga acatggtgag cccgctccgg 1920 agaggatgac ggatgaaatt tacgccaaca caacgacgat taagtcctac aagatttgta 1980 aggaattccc caacattgac attggcgctg cgggccactc caagatcatg tctgacgacg 2040 gcagcgccga agtcaatatt gaagtaattg attccaccga agctcacgtc aagttgttga 2100 aatctatttt tgatttctcg gccatcagag ggctgttgga tcgccccgac ttttccatgg 2160 tctacgacgc catgcacggt gtcaacgggc cgtacgtaaa aaaagtattc tgcgatattc 2220 tggggcagga cctctccgtc acactgaact gtgtccccaa ggacgacttc aacggaggcc 2280 atgccgaccc caacctcacg tacgccaaag agcttgttgc cgtcatgggg cttaatcgca 2340 agggcgaaaa gatcgatatg ggcggacgtc ctattcccag ctttggtgcg gccgccgacg 2400 gcgacggaga ccgcaacatg attctgggca cacagttttt tgtcagtccg tccgattcct 2460 tggcagtcat tgttgccaac gccgacacca ttccattctt ccgcacgcaa ggtggactca 2520 agggcgtcgc gcggtccatg ccaacgtccg gcgccgtcga tctcgtcgcc aaggacctga 2580 actacagttt gtttgaaaca cctacgggat ggaaatactt cgggaacctg atggattcca 2640 aagagctttt tgacggtgcc gaatacactc cgtttatttg tggggaagaa tcgttcggca 2700 caggctccga tcacattcgc gaaaaggacg gactttgggc cgtgctggct tggctcagca 2760 ttttggcgca cgccaatact aacagcctaa gtgacacact ggtgaccgtg gaagacattg 2820 tcaaggctca ttgggcaaag tacggacgca actactacag ccgctgggat ttcgagaaca 2880 tgaatgcgac caaggcgaac gccatgatgg acaagatgcg ggcggaaaca gacgcgaaca 2940 cgggcaagac ggtgggcaag tactcgatcg aaaagtccga cgactttgtg tacgtggatc 3000 ccgtggacgg ctcggtggcc aagaagcagg ggatgcggtt cctaatgacg gatggctcgc 3060 ggattatttt ccgtttgagt ggcacggcgg gcagtggcgc cacggtccgc atgtacatcg 3120 aacagtacga accgacgaag attgacatgg tggcttcaga ggctttggca gatttgattc 3180 gagtcgcact ggatttatct gacctcaagg gattcctcgg aactgaagaa ccaaccgtaa 3240 ttacgtaact gatgttcgag ctctggcaac acgtcctgct aggtctcagt gtggctaact 3300 aaacgagcca gccagaacag tttcctccgt ctgatatatg aatgatgtga ctcgctcagg 3360 aatcgattcg taattgtcga gtagagcaac ttaatagtgc aacaacgata gccctagtgc 3420 aaaatcctcg tctcgtttcg atgggttcat gcatcctaat gcaagctgaa tatttcgttg 3480 tctatccgag taatacaaag agaaaattcg gtatttggga tgagcagggg tgaaattttc 3540 Page 4 eolf-seql.txt gctatttggg aaaaatcaca ctgtttctaa gtgtttttat tttcgcggga aatactttct 3600 aagtaatctt ttt 3613

<210> 4

<211> 5922 <212> DNA <213> artificial sequence <220> <223> pCLS19745 (TALEN UGP)

<400> 4 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat tacccatacg atgttccaga ttacgctatc gatatcgccg accccattcg 2400 ttcgcgcaca ccaagtcctg cccgcgagct tctgcccgga ccccaacccg atggggttca 2460 gccgactgca gatcgtgggg tgtctccgcc tgccggcggc cccctggatg gcttgccggc 2520 tcggcggacg atgtcccgga cccggctgcc atctccccct gccccctcac ctgcgttctc 2580 ggcgggcagc ttcagtgacc tgttacgtca gttcgatccg tcacttttta atacatcgct 2640 ttttgattca ttgcctccct tcggcgctca ccatacagag gctgccacag gcgagtggga 2700 tgaggtgcaa tcgggtctgc gggcagccga cgccccccca cccaccatgc gcgtggctgt 2760 cactgccgcg cggcccccgc gcgccaagcc ggcgccgcga cgacgtgctg cgcaaccctc 2820 cgacgcttcg ccggcggcgc aggtggatct acgcacgctc ggctacagcc agcagcaaca 2880 ggagaagatc aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg 2940 ccacgggttt acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac 3000 cgtcgctgtc aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat 3060 cgttggcgtc ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc 3120 gggagagttg agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa 3180 acgtggcggc gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc 3240 Page 5 eolf-seql.txt cccgctcaac ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca 3300 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3360 ggagcaggtg gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca 3420 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 3480 cgccagccac gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3540 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 3600 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3660 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 3720 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt 3780 ggccatcgcc agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc 3840 ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg 3900 cggtggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 3960 cggcttgacc ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct 4020 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4080 ggtggtggcc atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct 4140 gttgccggtg ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag 4200 caataatggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4260 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca 4320 ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4380 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 4440 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 4500 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4560 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg 4620 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4680 gaccccccag caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac 4740 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt 4800 ggccatcgcc agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc 4860 tcgccctgat ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct 4920 cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc 4980 ccagctggtg aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta 5040 cgtgccccac gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat 5100 cctggagatg aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct 5160 gggcggctcc aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg 5220 cgtgatcgtg gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga 5280 cgaaatgcag aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga 5340 gtggtggaag gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca 5400 cttcaagggc aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg 5460 cgccgtgctg tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct 5520 gaccctggag gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg 5580 ataactcgag cgatcctcta gacgagctcc tcgagcctgc agcagctgaa gctttaagat 5640 ccaatggcaa ggaccaagtg ctggaacttg ttttgcttta gcagatctag atcgagctac 5700 ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa 5760 ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca cgaacagttg 5820 ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt 5880 ttcacagtca ggaataacac tagctcgtct tcatatcctg ca 5922

<210> 5 <211> 5940 <212> DNA <213> artificial sequence <220> <223> pCLS19749 (TALEN UGP) <400> 5 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 Page 6 eolf-seql.txt cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat aaggagaccg ccgctgccaa gttcgagaga cagcacatgg acagcatcga 2400 tatcgccgac cccattcgtt cgcgcacacc aagtcctgcc cgcgagcttc tgcccggacc 2460 ccaacccgat ggggttcagc cgactgcaga tcgtggggtg tctccgcctg ccggcggccc 2520 cctggatggc ttgccggctc ggcggacgat gtcccggacc cggctgccat ctccccctgc 2580 cccctcacct gcgttctcgg cgggcagctt cagtgacctg ttacgtcagt tcgatccgtc 2640 actttttaat acatcgcttt ttgattcatt gcctcccttc ggcgctcacc atacagaggc 2700 tgccacaggc gagtgggatg aggtgcaatc gggtctgcgg gcagccgacg cccccccacc 2760 caccatgcgc gtggctgtca ctgccgcgcg gcccccgcgc gccaagccgg cgccgcgacg 2820 acgtgctgcg caaccctccg acgcttcgcc ggcggcgcag gtggatctac gcacgctcgg 2880 ctacagccag cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca 2940 ccacgaggca ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca 3000 cccggcagcg ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga 3060 ggcgacacac gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga 3120 ggccttgctc acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca 3180 acttctcaag attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg 3240 caatgcactg acgggtgccc cgctcaactt gaccccggag caggtggtgg ccatcgccag 3300 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 3360 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 3420 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3480 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 3540 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3600 cgccagcaat aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3660 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg 3720 caagcaggcg ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt 3780 gaccccggag caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac 3840 ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt 3900 ggccatcgcc agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc 3960 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga 4020 tggcggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 4080 cggcttgacc ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct 4140 ggagacggtg caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4200 ggtggtggcc atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct 4260 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4320 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4380 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 4440 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4500 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 4560 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 4620 cgccagcaat attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct 4680 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 4740 Page 7 eolf-seql.txt caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4800 gacccctcag caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag 4860 cattgttgcc cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct 4920 cgtcgccttg gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg 4980 ggatcctatc agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt 5040 gaggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa 5100 cagcacccag gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg 5160 ctacaggggc aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg 5220 ctcccccatc gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct 5280 gcccatcggc caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa 5340 gcacatcaac cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt 5400 cctgttcgtg tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca 5460 catcaccaac tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat 5520 gatcaaggcc ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat 5580 caacttcgcg gccgactgat aactcgagcg atcctctaga cgagctcctc gagcctgcag 5640 cagctgaagc tttaagatcc aatggcaagg accaagtgct ggaacttgtt ttgctttagc 5700 agatctagat cgagctacct cgactttggc tgggacactt tcagtgagga caagaagctt 5760 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 5820 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 5880 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc atatcctgca 5940

<210> 6 <211> 50 <212> DNA <213> artificial sequence

<220> <223> UGPase target

<400> 6 tgccgccttc gagtcgacct atggtagtct cgtctcgggt gattccggaa 50

<210> 7 <211> 20 <212> DNA <213> artificial sequence

<220> <223> Screen TALEN_For

<400> 7 aatctcgcct attcatggtg 20

<210> 8

<211> 21 <212> DNA <213> artificial sequence

<220> <223> ScreenHA_Rev

<400> 8 taatctggaa catcgtatgg g 21

<210> 9 <211> 21 <212> DNA <213> artificial sequence

<220> Page 8 eolf-seql.txt <223> Screen Stag_Rev <400> 9 tgtctctcga acttggcagc g 21

<210> 10 <211> 63 <212> DNA <213> artificial sequence

<220> <221> misc_feature <222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40) <223> primers UGP_for; n is a or c or t or g <400> 10 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn gttgaatcgg aatcgctaac 60 tcg 63

<210> 11 <211> 52 <212> DNA <213> artificial sequence

<220> <223> primers UGP_Rev

<400> 11 cctatcccct gtgtgccttg gcagtctcag gacttgtttg gcggtcaaat cc 52

<210> 12 <211> 23 <212> DNA <213> artificial sequence

<220> <223> Deep seq UGP_for

<400> 12 gttgaatcgg aatcgctaac tcg 23

<210> 13

<211> 22 <212> DNA <213> artificial sequence

<220> <223> Deep seq UGP_Rev

<400> 13 gacttgtttg gcggtcaaat cc 22

<210> 14 <211> 747 <212> DNA <213> Phaeodactylum tricornutum

<220> Page 9 eolf-seql.txt <223> elongase putative protein (PHATRDRAFT_49867) <400> 14 atggaagcgc atccgttggt tcccattggc gcctgcctac tctacggact cttgatggtg 60 gcgggacagg cctactttcg cacacgcgaa ccactccggg cgcggacctc cctcgcggcc 120 tggaatctct ttctggccct cttttccctc gtcggcatgc tccggacctt tccccagctt 180 gtacacaacc tcgcgacgct cacgctccgg gaaaatctct gcgccaatcc gcaagccacc 240 tacggatccg gatccaccgg attgtgggta caactcttta ttctgtccaa attccctgaa 300 ctcattgata cagtattcat cattgtcaac aagaagaaac tcatcttctt acactggtac 360 catcacatta cggtcctcct ctactgctgg cacagttacg tcaccaaatc cccgccggga 420 attttctttg tcgtcatgaa ctacaccgtc cacgcctcca tgtacggata ctactttctc 480 atggccatcc gagcccgacc ccgttggctc aatcccatga ttgtcacgac tatgcaaata 540 tcgcaaatgg tcgtgggcgt cgccgtcacc ctccttggct tttactactc ggcacgtgcc 600 gccgaccacc aatcctgtcg aattaaacgg gaaaacaaca ccgccgcctt tgtcatgtac 660 ggatcctatc tatttctctt tctgcagttc tttgtgggac gctacgttgg cacccaatcc 720 ccagtcgcgt ccaaaaagac ggcctaa 747

<210> 15

<211> 5922 <212> DNA <213> artificial sequence <220> <223> pCLS19746 (TALEN Elongase) <400> 15 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat tacccatacg atgttccaga ttacgctatc gatatcgccg accccattcg 2400 Page 10 eolf-seql.txt ttcgcgcaca ccaagtcctg cccgcgagct tctgcccgga ccccaacccg atggggttca 2460 gccgactgca gatcgtgggg tgtctccgcc tgccggcggc cccctggatg gcttgccggc 2520 tcggcggacg atgtcccgga cccggctgcc atctccccct gccccctcac ctgcgttctc 2580 ggcgggcagc ttcagtgacc tgttacgtca gttcgatccg tcacttttta atacatcgct 2640 ttttgattca ttgcctccct tcggcgctca ccatacagag gctgccacag gcgagtggga 2700 tgaggtgcaa tcgggtctgc gggcagccga cgccccccca cccaccatgc gcgtggctgt 2760 cactgccgcg cggcccccgc gcgccaagcc ggcgccgcga cgacgtgctg cgcaaccctc 2820 cgacgcttcg ccggcggcgc aggtggatct acgcacgctc ggctacagcc agcagcaaca 2880 ggagaagatc aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg 2940 ccacgggttt acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac 3000 cgtcgctgtc aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat 3060 cgttggcgtc ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc 3120 gggagagttg agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa 3180 acgtggcggc gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc 3240 cccgctcaac ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 3300 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3360 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 3420 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3480 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3540 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg 3600 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3660 gaccccccag caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac 3720 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt 3780 ggccatcgcc agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc 3840 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga 3900 tggcggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 3960 cggcttgacc ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct 4020 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca 4080 ggtggtggcc atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct 4140 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4200 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4260 ggcccacggc ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca 4320 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4380 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 4440 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 4500 cgccagccac gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4560 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 4620 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4680 gaccccccag caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac 4740 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt 4800 ggccatcgcc agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc 4860 tcgccctgat ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct 4920 cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc 4980 ccagctggtg aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta 5040 cgtgccccac gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat 5100 cctggagatg aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct 5160 gggcggctcc aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg 5220 cgtgatcgtg gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga 5280 cgaaatgcag aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga 5340 gtggtggaag gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca 5400 cttcaagggc aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg 5460 cgccgtgctg tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct 5520 gaccctggag gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg 5580 ataactcgag cgatcctcta gacgagctcc tcgagcctgc agcagctgaa gctttaagat 5640 ccaatggcaa ggaccaagtg ctggaacttg ttttgcttta gcagatctag atcgagctac 5700 ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa 5760 ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca cgaacagttg 5820 ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt 5880 ttcacagtca ggaataacac tagctcgtct tcatatcctg ca 5922

<210> 16 <211> 5940 <212> DNA <213> artificial sequence

<220> Page 11 eolf-seql.txt <223> pCLS19750 (TALEN Elongase) <400> 16 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat aaggagaccg ccgctgccaa gttcgagaga cagcacatgg acagcatcga 2400 tatcgccgac cccattcgtt cgcgcacacc aagtcctgcc cgcgagcttc tgcccggacc 2460 ccaacccgat ggggttcagc cgactgcaga tcgtggggtg tctccgcctg ccggcggccc 2520 cctggatggc ttgccggctc ggcggacgat gtcccggacc cggctgccat ctccccctgc 2580 cccctcacct gcgttctcgg cgggcagctt cagtgacctg ttacgtcagt tcgatccgtc 2640 actttttaat acatcgcttt ttgattcatt gcctcccttc ggcgctcacc atacagaggc 2700 tgccacaggc gagtgggatg aggtgcaatc gggtctgcgg gcagccgacg cccccccacc 2760 caccatgcgc gtggctgtca ctgccgcgcg gcccccgcgc gccaagccgg cgccgcgacg 2820 acgtgctgcg caaccctccg acgcttcgcc ggcggcgcag gtggatctac gcacgctcgg 2880 ctacagccag cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca 2940 ccacgaggca ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca 3000 cccggcagcg ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga 3060 ggcgacacac gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga 3120 ggccttgctc acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca 3180 acttctcaag attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg 3240 caatgcactg acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag 3300 caatggcggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 3360 ggcccacggc ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca 3420 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3480 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 3540 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3600 cgccagcaat aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3660 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg 3720 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3780 gaccccggag caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac 3840 ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt 3900 Page 12 eolf-seql.txt ggccatcgcc agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc 3960 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat 4020 tggtggcaag caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca 4080 cggcttgacc ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct 4140 ggagacggtg caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca 4200 ggtggtggcc atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct 4260 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4320 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4380 ggcccacggc ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca 4440 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4500 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 4560 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 4620 cgccagcaat aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4680 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 4740 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4800 gacccctcag caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag 4860 cattgttgcc cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct 4920 cgtcgccttg gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg 4980 ggatcctatc agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt 5040 gaggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa 5100 cagcacccag gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg 5160 ctacaggggc aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg 5220 ctcccccatc gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct 5280 gcccatcggc caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa 5340 gcacatcaac cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt 5400 cctgttcgtg tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca 5460 catcaccaac tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat 5520 gatcaaggcc ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat 5580 caacttcgcg gccgactgat aactcgagcg atcctctaga cgagctcctc gagcctgcag 5640 cagctgaagc tttaagatcc aatggcaagg accaagtgct ggaacttgtt ttgctttagc 5700 agatctagat cgagctacct cgactttggc tgggacactt tcagtgagga caagaagctt 5760 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 5820 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 5880 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc atatcctgca 5940

<210> 17

<211> 49 <212> DNA <213> artificial sequence

<220> <223> Elongase target

<400> 17 tcttttccct cgtcggcatg ctccggacct ttccccagct tgtacacaa 49

<210> 18

<211> 59 <212> DNA <213> artificial sequence <220> <221> misc_feature <222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40) <223> primer Elongase_For ; n is a or c or t or g

<400> 18 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn aagcgcatcc gttggttcc 59

<210> 19

<211> 52 Page 13 eolf-seql.txt <212> DNA <213> artificial sequence

<220> <223> primer Elongase_Rev

<400> 19 cctatcccct gtgtgccttg gcagtctcag tcaatgagtt cactggaaag gg 52

<210> 20

<211> 19 <212> DNA <213> artificial sequence

<220> <223> Deep seq Elongase_For

<400> 20 aagcgcatcc gttggttcc 19

<210> 21

<211> 22 <212> DNA <213> artificial sequence

<220> <223> Deep seq Elongase_Rev <400> 21 tcaatgagtt cactggaaag gg 22

<210> 22

<211> 1161 <212> DNA <213> Phaeodactylum tricornutum

<220> <223> hypothetical protein : glycerol-3-phodsphate deshydrogenase (PHATR_36821)

<400> 22 atggactcaa atcctcaaaa tagttctgac caactcgata aagtatgcat catcggtagc 60 ggtaactggg gaagtgccat tgcgacccta gttggtcgca actgcgagcg cttgcccttt 120 ttcgaatcgc aggtcaacat gtgggtcttt gaggaaatgg ttgaattgga agatggttcc 180 caaaagaagc tcaccgaaat catcaactct cgccacgaaa acgttaagta cctcccaggc 240 attcccctcc cttccaacgt tgttgcgact cctgatctag cagaagcctg tcgcgatgcc 300 acgctcttga tctttgtcct accccaccaa ttcttaccgc gactacttcc cgtcattcgc 360 gagtcggcgc acccaacctg tcggggggtt agtctcatca aagggcttga cttcgactcg 420 gaacgcaaac ttccaatcct tatttccaac acaatcgctg acgccatggg acctgaattt 480 caatgcggcg ttctgatggg agcgaacgtt gcctccgagg ttgctctggg tcaaatgtgc 540 gagtccacct tggcgtctcc ctttggtcca ccagcagatg agctgacacg tctcgtcttt 600 gacgctccct ccttccgagt gcagcacgtg ccagacgttg cgggtgccga agtctgcggt 660 gcgcttaaga acgtagttgc tctcggcgca ggttttgttg atggcgttgg actcggaagc 720 aatactaagg cggctctgct tagagtggga cttcgagaga tggccaagtt ttgccacatg 780 ttctttgacg gcgttcaaga taataccttt acgcagagct gtggcatggc agatttaatc 840 acgacatgct acggtggaag gaatcgcaaa tgtgcggaag cttttgcgaa ggaacgtctt 900 ggatcggacg ggctctgcga cgaggctatg gcgtgtgaac aaaagtggga gaagattgaa 960 gccaaacttc tcaacggcca aaagctgcaa ggaactctaa cggcgaaaga agttcacgcc 1020 atacttgact ctcgaggggt ccttaacgca tttcctctaa tcaaaacgat ccatgagatt 1080 tcttttaaag ggaaacctgt acaacagatt gtggatggta ttattgatac gaacgagcac 1140 ggagctagct cgcacttgta a 1161

Page 14 eolf-seql.txt <210> 23 <211> 5922 <212> DNA <213> artificial sequence

<220> <223> pCLS23159 (TALEN G3PDH) <400> 23 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat tacccatacg atgttccaga ttacgctatc gatatcgccg accccattcg 2400 ttcgcgcaca ccaagtcctg cccgcgagct tctgcccgga ccccaacccg atggggttca 2460 gccgactgca gatcgtgggg tgtctccgcc tgccggcggc cccctggatg gcttgccggc 2520 tcggcggacg atgtcccgga cccggctgcc atctccccct gccccctcac ctgcgttctc 2580 ggcgggcagc ttcagtgacc tgttacgtca gttcgatccg tcacttttta atacatcgct 2640 ttttgattca ttgcctccct tcggcgctca ccatacagag gctgccacag gcgagtggga 2700 tgaggtgcaa tcgggtctgc gggcagccga cgccccccca cccaccatgc gcgtggctgt 2760 cactgccgcg cggcccccgc gcgccaagcc ggcgccgcga cgacgtgctg cgcaaccctc 2820 cgacgcttcg ccggcggcgc aggtggatct acgcacgctc ggctacagcc agcagcaaca 2880 ggagaagatc aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg 2940 ccacgggttt acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac 3000 cgtcgctgtc aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat 3060 cgttggcgtc ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc 3120 gggagagttg agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa 3180 acgtggcggc gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc 3240 cccgctcaac ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 3300 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3360 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 3420 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3480 Page 15 eolf-seql.txt cgccagcaat aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3540 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg 3600 caagcaggcg ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt 3660 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 3720 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt 3780 ggccatcgcc agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc 3840 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat 3900 tggtggcaag caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca 3960 cggcttgacc ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct 4020 ggagacggtg caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4080 ggtggtggcc atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct 4140 gttgccggtg ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag 4200 caatggcggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4260 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 4320 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4380 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 4440 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 4500 cgccagcaat attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct 4560 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg 4620 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4680 gaccccggag caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac 4740 ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt 4800 ggccatcgcc agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc 4860 tcgccctgat ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct 4920 cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc 4980 ccagctggtg aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta 5040 cgtgccccac gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat 5100 cctggagatg aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct 5160 gggcggctcc aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg 5220 cgtgatcgtg gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga 5280 cgaaatgcag aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga 5340 gtggtggaag gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca 5400 cttcaagggc aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg 5460 cgccgtgctg tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct 5520 gaccctggag gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg 5580 ataactcgag cgatcctcta gacgagctcc tcgagcctgc agcagctgaa gctttaagat 5640 ccaatggcaa ggaccaagtg ctggaacttg ttttgcttta gcagatctag atcgagctac 5700 ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa 5760 ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca cgaacagttg 5820 ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt 5880 ttcacagtca ggaataacac tagctcgtct tcatatcctg ca 5922

<210> 24

<211> 5940 <212> DNA <213> artificial sequence <220> <223> pCLS23163 (TALEN G3PDH) <400> 24 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 Page 16 eolf-seql.txt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat aaggagaccg ccgctgccaa gttcgagaga cagcacatgg acagcatcga 2400 tatcgccgac cccattcgtt cgcgcacacc aagtcctgcc cgcgagcttc tgcccggacc 2460 ccaacccgat ggggttcagc cgactgcaga tcgtggggtg tctccgcctg ccggcggccc 2520 cctggatggc ttgccggctc ggcggacgat gtcccggacc cggctgccat ctccccctgc 2580 cccctcacct gcgttctcgg cgggcagctt cagtgacctg ttacgtcagt tcgatccgtc 2640 actttttaat acatcgcttt ttgattcatt gcctcccttc ggcgctcacc atacagaggc 2700 tgccacaggc gagtgggatg aggtgcaatc gggtctgcgg gcagccgacg cccccccacc 2760 caccatgcgc gtggctgtca ctgccgcgcg gcccccgcgc gccaagccgg cgccgcgacg 2820 acgtgctgcg caaccctccg acgcttcgcc ggcggcgcag gtggatctac gcacgctcgg 2880 ctacagccag cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca 2940 ccacgaggca ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca 3000 cccggcagcg ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga 3060 ggcgacacac gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga 3120 ggccttgctc acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca 3180 acttctcaag attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg 3240 caatgcactg acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag 3300 caatggcggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 3360 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 3420 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3480 ggagcaggtg gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca 3540 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 3600 cgccagccac gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3660 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg 3720 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3780 gaccccggag caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac 3840 ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt 3900 ggccatcgcc agcaataatg gtggcaagca ggcgctggag acggtccagc ggctgttgcc 3960 ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg 4020 cggtggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 4080 cggcttgacc ccccagcagg tggtggccat cgccagcaat ggcggtggca agcaggcgct 4140 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4200 ggtggtggcc atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct 4260 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4320 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4380 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 4440 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4500 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 4560 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 4620 cgccagccac gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4680 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg 4740 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4800 gacccctcag caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag 4860 cattgttgcc cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct 4920 cgtcgccttg gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg 4980 Page 17 eolf-seql.txt ggatcctatc agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt 5040 gaggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa 5100 cagcacccag gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg 5160 ctacaggggc aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg 5220 ctcccccatc gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct 5280 gcccatcggc caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa 5340 gcacatcaac cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt 5400 cctgttcgtg tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca 5460 catcaccaac tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat 5520 gatcaaggcc ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat 5580 caacttcgcg gccgactgat aactcgagcg atcctctaga cgagctcctc gagcctgcag 5640 cagctgaagc tttaagatcc aatggcaagg accaagtgct ggaacttgtt ttgctttagc 5700 agatctagat cgagctacct cgactttggc tgggacactt tcagtgagga caagaagctt 5760 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 5820 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 5880 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc atatcctgca 5940

<210> 25

<211> 50 <212> DNA <213> artificial sequence <220> <223> G3PDH target <400> 25 ttctgaccaa ctcgataaag tatgcatcat cggtagcggt aactggggaa 50

<210> 26

<211> 26 <212> DNA <213> artificial sequence <220> <223> ScreenHA_For <400> 26 acccatacga tgttccagat tacgct 26

<210> 27

<211> 24 <212> DNA <213> artificial sequence

<220> <223> Screen TALEN_Rev <400> 27 aatcttgaga agttggcctg tgtc 24

<210> 28 <211> 62 <212> DNA <213> artificial sequence <220> <221> misc_feature <222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40) <223> primer G3PDH_For ; n is a or c or t or g

Page 18 eolf-seql.txt <400> 28 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn tctgctactg ctcatccgca 60 cc 62

<210> 29 <211> 53 <212> DNA <213> artificial sequence

<220> <223> primer G3PDH_Rev <400> 29 cctatcccct gtgtgccttg gcagtctcag tcgcgacagg cttctgctag atc 53

<210> 30 <211> 1308 <212> DNA <213> Phaeodactylum tricornutum

<220> <223> precursor of desaturase omega-3 desaturase (PTD15) (PHATRDRAFT_41570)

<400> 30 atgaagcttc atatcgcacc acctcttatt atctcggcct acgtgttttc tgtatccatt 60 ttccacaaca ctgttaatgc cttttcgttg cgcataccga gtacccacag gactgttttc 120 cttccgcaag tgacgttgaa tgccaaaaga tggatggtag caacgggggt agaaacaaac 180 gctgctgtgg caactccaga aaatgacgaa atccatcctc gacgggattg gactcacgac 240 gagccgccca agttgagcga agtgaagcgc atgcttcccc aagaagcctt ccacattgat 300 acagcaacgt cactttttta ttttgcggtg gattttatcg ctgtagcatc cactatggga 360 tttctgaact ccgtcgtctc atccgatatc tacctttcct tccctatctg gggcaagttc 420 ttggctgtag cccctttaca gattttgacc ggttttgcga tgtggtgcat gtggtgtatc 480 ggccacgatg ccggtcacac tactgtatcc aaagaccggc ggttcggcgc tcttattaat 540 agggtagttg gcgaagtggc gcattccgcg atttgcttaa cgcctttcgt tccctgggcc 600 aagtctcatc tgaagcatca catgggacac aaccacttga cgcgtgacta ctcgcatcaa 660 tggtttatcc gagaagaacg agagtcgcta catccactta ttcaactgag tcatgcgacg 720 cgaaatttac agttaccgat actttacctc gtttatctct tatttggggt tcccgatgga 780 ggacacgtcg ttttttacgg acgcatgtgg gagcagtcta ccgcgaagga aaaggccgat 840 gccgcggtgt ccgttatcgt atcccttgtc acggccggtt ctctgtggat caatatgggt 900 ttggcaaatt tcttcgttgt ttgcatggtc ccgtggttgg tcctgtcatt ttggctcttc 960 atggtcacct atctacagca ccactccgac gatggtttac tctatacgga cgagacctgg 1020 agctttgaac ggggcgcctt tcaaactgtt gatcgtgatt atggaacgtg gatcaatcgc 1080 atgtcacatc acatgatgga tgggcatttg gtccatcacc tgtttttcac tcgggttccg 1140 cattacaggc tcgaagaagc gacgaaatcc ttatatgcag tcatggctgc gcgagggcag 1200 tcacacctga ttaaaacgat tgatacgccc gactttacgc aggagattgc caaacaattc 1260 gacaaaaact ggttctttgt caacgaaaat cagattgtac gcaagtaa 1308

<210> 31 <211> 5922 <212> DNA <213> artificial sequence <220> <223> pCLS23158 (TALEN Omega3 desaturase) <400> 31 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 Page 19 eolf-seql.txt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat tacccatacg atgttccaga ttacgctatc gatatcgccg accccattcg 2400 ttcgcgcaca ccaagtcctg cccgcgagct tctgcccgga ccccaacccg atggggttca 2460 gccgactgca gatcgtgggg tgtctccgcc tgccggcggc cccctggatg gcttgccggc 2520 tcggcggacg atgtcccgga cccggctgcc atctccccct gccccctcac ctgcgttctc 2580 ggcgggcagc ttcagtgacc tgttacgtca gttcgatccg tcacttttta atacatcgct 2640 ttttgattca ttgcctccct tcggcgctca ccatacagag gctgccacag gcgagtggga 2700 tgaggtgcaa tcgggtctgc gggcagccga cgccccccca cccaccatgc gcgtggctgt 2760 cactgccgcg cggcccccgc gcgccaagcc ggcgccgcga cgacgtgctg cgcaaccctc 2820 cgacgcttcg ccggcggcgc aggtggatct acgcacgctc ggctacagcc agcagcaaca 2880 ggagaagatc aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg 2940 ccacgggttt acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac 3000 cgtcgctgtc aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat 3060 cgttggcgtc ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc 3120 gggagagttg agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa 3180 acgtggcggc gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc 3240 cccgctcaac ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca 3300 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3360 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 3420 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3480 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3540 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg 3600 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3660 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 3720 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt 3780 ggccatcgcc agcaatattg gtggcaagca ggcgctggag acggtgcagg cgctgttgcc 3840 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga 3900 tggcggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 3960 cggcttgacc ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct 4020 ggagacggtg caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4080 ggtggtggcc atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct 4140 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4200 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4260 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca 4320 ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4380 Page 20 eolf-seql.txt ggagcaggtg gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca 4440 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 4500 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4560 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 4620 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4680 gaccccccag caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac 4740 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt 4800 ggccatcgcc agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc 4860 tcgccctgat ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct 4920 cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc 4980 ccagctggtg aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta 5040 cgtgccccac gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat 5100 cctggagatg aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct 5160 gggcggctcc aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg 5220 cgtgatcgtg gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga 5280 cgaaatgcag aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga 5340 gtggtggaag gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca 5400 cttcaagggc aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg 5460 cgccgtgctg tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct 5520 gaccctggag gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg 5580 ataactcgag cgatcctcta gacgagctcc tcgagcctgc agcagctgaa gctttaagat 5640 ccaatggcaa ggaccaagtg ctggaacttg ttttgcttta gcagatctag atcgagctac 5700 ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa 5760 ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca cgaacagttg 5820 ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt 5880 ttcacagtca ggaataacac tagctcgtct tcatatcctg ca 5922

<210> 32

<211> 5940 <212> DNA <213> artificial sequence

<220> <223> pCLS23162 (TALEN Omega3 desaturase) <400> 32 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 Page 21 eolf-seql.txt caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat aaggagaccg ccgctgccaa gttcgagaga cagcacatgg acagcatcga 2400 tatcgccgac cccattcgtt cgcgcacacc aagtcctgcc cgcgagcttc tgcccggacc 2460 ccaacccgat ggggttcagc cgactgcaga tcgtggggtg tctccgcctg ccggcggccc 2520 cctggatggc ttgccggctc ggcggacgat gtcccggacc cggctgccat ctccccctgc 2580 cccctcacct gcgttctcgg cgggcagctt cagtgacctg ttacgtcagt tcgatccgtc 2640 actttttaat acatcgcttt ttgattcatt gcctcccttc ggcgctcacc atacagaggc 2700 tgccacaggc gagtgggatg aggtgcaatc gggtctgcgg gcagccgacg cccccccacc 2760 caccatgcgc gtggctgtca ctgccgcgcg gcccccgcgc gccaagccgg cgccgcgacg 2820 acgtgctgcg caaccctccg acgcttcgcc ggcggcgcag gtggatctac gcacgctcgg 2880 ctacagccag cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca 2940 ccacgaggca ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca 3000 cccggcagcg ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga 3060 ggcgacacac gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga 3120 ggccttgctc acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca 3180 acttctcaag attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg 3240 caatgcactg acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag 3300 caataatggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 3360 ggcccacggc ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca 3420 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3480 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 3540 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3600 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3660 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg 3720 caagcaggcg ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt 3780 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 3840 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt 3900 ggccatcgcc agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc 3960 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga 4020 tggcggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 4080 cggcttgacc ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct 4140 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca 4200 ggtggtggcc atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct 4260 gttgccggtg ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag 4320 caatggcggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4380 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca 4440 ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4500 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 4560 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 4620 cgccagcaat aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4680 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg 4740 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4800 gacccctcag caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag 4860 cattgttgcc cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct 4920 cgtcgccttg gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg 4980 ggatcctatc agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt 5040 gaggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa 5100 cagcacccag gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg 5160 ctacaggggc aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg 5220 ctcccccatc gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct 5280 gcccatcggc caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa 5340 gcacatcaac cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt 5400 cctgttcgtg tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca 5460 catcaccaac tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat 5520 gatcaaggcc ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat 5580 caacttcgcg gccgactgat aactcgagcg atcctctaga cgagctcctc gagcctgcag 5640 cagctgaagc tttaagatcc aatggcaagg accaagtgct ggaacttgtt ttgctttagc 5700 agatctagat cgagctacct cgactttggc tgggacactt tcagtgagga caagaagctt 5760 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 5820 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 5880 Page 22 eolf-seql.txt cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc atatcctgca 5940

<210> 33 <211> 49 <212> DNA <213> artificial sequence <220> <223> Omega3 desaturase target

<400> 33 ttttccacaa cactgttaat gccttttcgt tgcgcatacc gagtaccca 49

<210> 34 <211> 61 <212> DNA <213> artificial sequence <220> <221> misc_feature <222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40) <223> Deep seq Omega3 desaturase_For ; n is a or c or t or g <400> 34 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn gcgtgtgctc acctgttgtc 60 c 61

<210> 35

<211> 52 <212> DNA <213> artificial sequence

<220> <223> Deep seqOmega3 desaturase_Rev

<400> 35 cctatcccct gtgtgccttg gcagtctcag aagcatgcgc ttcacttcgc tc 52

<210> 36 <211> 897 <212> DNA <213> Phaeodactylum tricornutum

<220> <223> hypothetical protein : palmitoyl-protein thioesterase (PHATR_10454)

<400> 36 tctgaaaaca agcaattgcc ggtggtcttt gcccatggga tgggagattc gtgctttaat 60 tctggcatgc aacacgttgc gcagctggcc tccgaatggc tcagtgagga ctttggtccg 120 gacagatcga atgtgtatag cgtatgcgtt ccgaccggtg cgactcaagc agaagatacc 180 aagaacggtt actttctgag catggatgct tcggtggaag tctttgcgga aggtgttagg 240 gcggacccac gactgagcga tggctttcac gccattgggt tttcgcaggg caacaacgtc 300 attcggggct acattgccaa acataacacg cctaccgttg acacatttat atccatcaat 360 ggggtgaacg cagggatcgg tgctgtgccg tattgtcgtc ctagtgaaac tgccatgggt 420 gctgtgcgac tgggtggaat gtgcgattta ctcatggaac aggcctcgcg gagtgcctac 480 actgaatttg cacaagagca ttcctttcaa gccaactact ggcgcgatcc acggccaact 540 gcctttccgc tctaccaaaa gtacggacag ctcgctgctt ggaataacga agcgggacta 600 gtgaacgaaa ctctgaaaac gaactggggt aagacctccg ctttcgtgtg ggtgttggcc 660 accgaagatg gattggtgtg gcccaaagaa ggagagcaat gggggcagcc ggattcgaaa 720 gatccttttc atgtgatctt atccataaac gaaacgacct ggtacaaaga ggacttgttt 780 Page 23 eolf-seql.txt ggcctccgaa ctgcaaatga attgggaaag aattatttcg aatcgttcga gggggaccat 840 ttgcagtttg aatctgccga tctggaacga tgggtaaaga cctacctcaa gaactag 897

<210> 37

<211> 5922 <212> DNA <213> artificial sequence <220> <223> pCLS19744 (TALEN PPT)

<400> 37 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat tacccatacg atgttccaga ttacgctatc gatatcgccg accccattcg 2400 ttcgcgcaca ccaagtcctg cccgcgagct tctgcccgga ccccaacccg atggggttca 2460 gccgactgca gatcgtgggg tgtctccgcc tgccggcggc cccctggatg gcttgccggc 2520 tcggcggacg atgtcccgga cccggctgcc atctccccct gccccctcac ctgcgttctc 2580 ggcgggcagc ttcagtgacc tgttacgtca gttcgatccg tcacttttta atacatcgct 2640 ttttgattca ttgcctccct tcggcgctca ccatacagag gctgccacag gcgagtggga 2700 tgaggtgcaa tcgggtctgc gggcagccga cgccccccca cccaccatgc gcgtggctgt 2760 cactgccgcg cggcccccgc gcgccaagcc ggcgccgcga cgacgtgctg cgcaaccctc 2820 cgacgcttcg ccggcggcgc aggtggatct acgcacgctc ggctacagcc agcagcaaca 2880 ggagaagatc aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg 2940 ccacgggttt acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac 3000 cgtcgctgtc aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat 3060 cgttggcgtc ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc 3120 gggagagttg agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa 3180 acgtggcggc gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc 3240 Page 24 eolf-seql.txt cccgctcaac ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca 3300 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3360 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 3420 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3480 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3540 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg 3600 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3660 gaccccccag caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac 3720 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt 3780 ggccatcgcc agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc 3840 ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaatgg 3900 cggtggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 3960 cggcttgacc ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct 4020 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4080 ggtggtggcc atcgccagcc acgatggcgg caagcaggcg ctggagacgg tccagcggct 4140 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4200 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4260 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 4320 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4380 ggagcaggtg gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca 4440 ggcgctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 4500 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4560 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 4620 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4680 gaccccccag caggtggtgg ccatcgccag caataatggt ggcaagcagg cgctggagac 4740 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt 4800 ggccatcgcc agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc 4860 tcgccctgat ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct 4920 cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc 4980 ccagctggtg aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta 5040 cgtgccccac gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat 5100 cctggagatg aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct 5160 gggcggctcc aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg 5220 cgtgatcgtg gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga 5280 cgaaatgcag aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga 5340 gtggtggaag gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca 5400 cttcaagggc aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg 5460 cgccgtgctg tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct 5520 gaccctggag gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg 5580 ataactcgag cgatcctcta gacgagctcc tcgagcctgc agcagctgaa gctttaagat 5640 ccaatggcaa ggaccaagtg ctggaacttg ttttgcttta gcagatctag atcgagctac 5700 ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa 5760 ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca cgaacagttg 5820 ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt 5880 ttcacagtca ggaataacac tagctcgtct tcatatcctg ca 5922

<210> 38 <211> 5940 <212> DNA <213> artificial sequence <220> <223> pCLS19748 (TALEN PPT) <400> 38 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 Page 25 eolf-seql.txt cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat aaggagaccg ccgctgccaa gttcgagaga cagcacatgg acagcatcga 2400 tatcgccgac cccattcgtt cgcgcacacc aagtcctgcc cgcgagcttc tgcccggacc 2460 ccaacccgat ggggttcagc cgactgcaga tcgtggggtg tctccgcctg ccggcggccc 2520 cctggatggc ttgccggctc ggcggacgat gtcccggacc cggctgccat ctccccctgc 2580 cccctcacct gcgttctcgg cgggcagctt cagtgacctg ttacgtcagt tcgatccgtc 2640 actttttaat acatcgcttt ttgattcatt gcctcccttc ggcgctcacc atacagaggc 2700 tgccacaggc gagtgggatg aggtgcaatc gggtctgcgg gcagccgacg cccccccacc 2760 caccatgcgc gtggctgtca ctgccgcgcg gcccccgcgc gccaagccgg cgccgcgacg 2820 acgtgctgcg caaccctccg acgcttcgcc ggcggcgcag gtggatctac gcacgctcgg 2880 ctacagccag cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca 2940 ccacgaggca ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca 3000 cccggcagcg ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga 3060 ggcgacacac gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga 3120 ggccttgctc acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca 3180 acttctcaag attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg 3240 caatgcactg acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag 3300 caataatggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 3360 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 3420 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3480 ggagcaggtg gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca 3540 ggcgctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3600 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3660 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 3720 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3780 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 3840 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt 3900 ggccatcgcc agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc 3960 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagcaatat 4020 tggtggcaag caggcgctgg agacggtgca ggcgctgttg ccggtgctgt gccaggccca 4080 cggcttgacc ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct 4140 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4200 ggtggtggcc atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct 4260 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4320 caatattggt ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca 4380 ggcccacggc ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca 4440 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4500 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 4560 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 4620 cgccagcaat attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct 4680 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg 4740 Page 26 eolf-seql.txt caagcaggcg ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt 4800 gacccctcag caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag 4860 cattgttgcc cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct 4920 cgtcgccttg gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg 4980 ggatcctatc agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt 5040 gaggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa 5100 cagcacccag gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg 5160 ctacaggggc aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg 5220 ctcccccatc gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct 5280 gcccatcggc caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa 5340 gcacatcaac cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt 5400 cctgttcgtg tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca 5460 catcaccaac tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat 5520 gatcaaggcc ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat 5580 caacttcgcg gccgactgat aactcgagcg atcctctaga cgagctcctc gagcctgcag 5640 cagctgaagc tttaagatcc aatggcaagg accaagtgct ggaacttgtt ttgctttagc 5700 agatctagat cgagctacct cgactttggc tgggacactt tcagtgagga caagaagctt 5760 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 5820 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 5880 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc atatcctgca 5940

<210> 39 <211> 50 <212> DNA <213> artificial sequence

<220> <223> PPT target

<400> 39 tggtctttgc ccatgggatg ggagattcgt gctttaattc tggcatgcaa 50

<210> 40 <211> 62 <212> DNA <213> artificial sequence

<220> <221> misc_feature <222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40) <223> Deep seq PPT_For ; n is a or c or t or g

<400> 40 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn gaagaacagt cgcacctggt 60 gc 62

<210> 41 <211> 50 <212> DNA <213> artificial sequence

<220> <223> Deep seq PPT_Rev <400> 41 cctatcccct gtgtgccttg gcagtctcag tccgccctaa caccttccgc 50

<210> 42 <211> 933 <212> DNA Page 27 eolf-seql.txt <213> Phaeodactylum tricornutum <220> <223> enoyl-acp reductase (PHATRDRAFT_10068) <400> 42 atggcagcgc aggtcgacct caaaggcaag gtagcctttg tggctggtgt tgccgattcc 60 actggttacg gctgggcgat cgccaaagct ttggccgaag caggagccac aatcattgtc 120 ggaacgtggc ctccggtact caagatcttc caaatgggtt tgaaaaaggg acagttcaac 180 gaggactcca cactcgcgga tggttcccta atgacgatcg aaaaggtgta tcccctcgat 240 gccgtctttg atgccccaga cgacgtcccg gatgagatta aggaaaataa gcgttacgct 300 ggattggacg gatacaccat ttccgaagta gccaaagccg tcgaagccga ttacggaaaa 360 atcgatatcc tcgttcattc cctcgccaac ggtcccgaag tcaccaagcc cctcctcgaa 420 acgactcgca aaggatacct cgctgcttct tccgcctccg catactcggc agtctcgctc 480 ctgcaaaaat tcggccccat catgaacgaa ggcggcgcca tgctttcgtt gacgtacatt 540 gcctccgaaa aggtcattcc cggttacggc ggtggcatgt cctccgccaa ggctcagctg 600 gaaagcgaca cccgcaccct cgcctacgaa gccggtcgca agtggggtat tcgcgtcaac 660 accatttccg ccggtcctct caaatcccgg gcggcctccg caattggcaa agagcccggt 720 cagaaaacct ttatcgaata cgccattgat tattccaagg ccaacgcgcc gctcgaacag 780 gatttgtaca gcgacgatgt cggtaacgcc agtctctttt tgaccagccc catggcccga 840 accgttaccg gtgttaccct gtacgtggac aatggcctgc attctatggg tatggccttg 900 gatagcaaag cgatggaagg ctcgcgcgag taa 933

<210> 43

<211> 5922 <212> DNA <213> artificial sequence

<220> <223> pCLS23157 (TALEN Enoyl_ACP_reductase)

<400> 43 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 Page 28 eolf-seql.txt tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat tacccatacg atgttccaga ttacgctatc gatatcgccg accccattcg 2400 ttcgcgcaca ccaagtcctg cccgcgagct tctgcccgga ccccaacccg atggggttca 2460 gccgactgca gatcgtgggg tgtctccgcc tgccggcggc cccctggatg gcttgccggc 2520 tcggcggacg atgtcccgga cccggctgcc atctccccct gccccctcac ctgcgttctc 2580 ggcgggcagc ttcagtgacc tgttacgtca gttcgatccg tcacttttta atacatcgct 2640 ttttgattca ttgcctccct tcggcgctca ccatacagag gctgccacag gcgagtggga 2700 tgaggtgcaa tcgggtctgc gggcagccga cgccccccca cccaccatgc gcgtggctgt 2760 cactgccgcg cggcccccgc gcgccaagcc ggcgccgcga cgacgtgctg cgcaaccctc 2820 cgacgcttcg ccggcggcgc aggtggatct acgcacgctc ggctacagcc agcagcaaca 2880 ggagaagatc aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg 2940 ccacgggttt acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac 3000 cgtcgctgtc aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat 3060 cgttggcgtc ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc 3120 gggagagttg agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa 3180 acgtggcggc gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc 3240 cccgctcaac ttgacccccc agcaggtggt ggccatcgcc agcaatggcg gtggcaagca 3300 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3360 ggagcaggtg gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca 3420 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 3480 cgccagccac gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3540 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg 3600 caagcaggcg ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt 3660 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 3720 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt 3780 ggccatcgcc agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc 3840 ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa 3900 tggtggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 3960 cggcttgacc ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct 4020 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca 4080 ggtggtggcc atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct 4140 gttgccggtg ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag 4200 caatggcggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4260 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca 4320 ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4380 ggagcaggtg gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca 4440 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 4500 cgccagcaat aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4560 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 4620 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4680 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 4740 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt 4800 ggccatcgcc agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc 4860 tcgccctgat ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct 4920 cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc 4980 ccagctggtg aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta 5040 cgtgccccac gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat 5100 cctggagatg aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct 5160 gggcggctcc aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg 5220 cgtgatcgtg gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga 5280 cgaaatgcag aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga 5340 gtggtggaag gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca 5400 cttcaagggc aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg 5460 cgccgtgctg tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct 5520 gaccctggag gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg 5580 ataactcgag cgatcctcta gacgagctcc tcgagcctgc agcagctgaa gctttaagat 5640 ccaatggcaa ggaccaagtg ctggaacttg ttttgcttta gcagatctag atcgagctac 5700 ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa 5760 ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca cgaacagttg 5820 ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt 5880 ttcacagtca ggaataacac tagctcgtct tcatatcctg ca 5922

<210> 44 Page 29 eolf-seql.txt <211> 5940 <212> DNA <213> artificial sequence <220> <223> pCLS23161 (TALEN Enoyl_ACP_reductase) <400> 44 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat aaggagaccg ccgctgccaa gttcgagaga cagcacatgg acagcatcga 2400 tatcgccgac cccattcgtt cgcgcacacc aagtcctgcc cgcgagcttc tgcccggacc 2460 ccaacccgat ggggttcagc cgactgcaga tcgtggggtg tctccgcctg ccggcggccc 2520 cctggatggc ttgccggctc ggcggacgat gtcccggacc cggctgccat ctccccctgc 2580 cccctcacct gcgttctcgg cgggcagctt cagtgacctg ttacgtcagt tcgatccgtc 2640 actttttaat acatcgcttt ttgattcatt gcctcccttc ggcgctcacc atacagaggc 2700 tgccacaggc gagtgggatg aggtgcaatc gggtctgcgg gcagccgacg cccccccacc 2760 caccatgcgc gtggctgtca ctgccgcgcg gcccccgcgc gccaagccgg cgccgcgacg 2820 acgtgctgcg caaccctccg acgcttcgcc ggcggcgcag gtggatctac gcacgctcgg 2880 ctacagccag cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca 2940 ccacgaggca ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca 3000 cccggcagcg ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga 3060 ggcgacacac gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga 3120 ggccttgctc acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca 3180 acttctcaag attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg 3240 caatgcactg acgggtgccc cgctcaactt gaccccggag caggtggtgg ccatcgccag 3300 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 3360 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 3420 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3480 ccagcaggtg gtggccatcg ccagcaatgg cggtggcaag caggcgctgg agacggtcca 3540 Page 30 eolf-seql.txt gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3600 cgccagcaat aatggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3660 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg 3720 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3780 gaccccccag caggtggtgg ccatcgccag caatggcggt ggcaagcagg cgctggagac 3840 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt 3900 ggccatcgcc agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc 3960 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga 4020 tggcggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 4080 cggcttgacc ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct 4140 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca 4200 ggtggtggcc atcgccagca ataatggtgg caagcaggcg ctggagacgg tccagcggct 4260 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4320 ccacgatggc ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4380 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agccacgatg gcggcaagca 4440 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4500 ggagcaggtg gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca 4560 ggcgctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 4620 cgccagcaat attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct 4680 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg 4740 caagcaggcg ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt 4800 gacccctcag caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag 4860 cattgttgcc cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct 4920 cgtcgccttg gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg 4980 ggatcctatc agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt 5040 gaggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa 5100 cagcacccag gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg 5160 ctacaggggc aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg 5220 ctcccccatc gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct 5280 gcccatcggc caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa 5340 gcacatcaac cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt 5400 cctgttcgtg tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca 5460 catcaccaac tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat 5520 gatcaaggcc ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat 5580 caacttcgcg gccgactgat aactcgagcg atcctctaga cgagctcctc gagcctgcag 5640 cagctgaagc tttaagatcc aatggcaagg accaagtgct ggaacttgtt ttgctttagc 5700 agatctagat cgagctacct cgactttggc tgggacactt tcagtgagga caagaagctt 5760 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 5820 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 5880 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc atatcctgca 5940

<210> 45

<211> 58 <212> DNA <213> artificial sequence <220> <223> Enoyl_ACP_reductase target

<400> 45 tgttgccgat tccactggtt acggctgggc gatcgccaaa gctttggccg aagcagga 58

<210> 46

<211> 63 <212> DNA <213> artificial sequence

<220> <221> misc_feature <222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40) <223> Deep seq Enoyl_ACP_reductase_For ; n is a or c or t or g <400> 46 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn ggactgtttc gctacggtac 60 Page 31 eolf-seql.txt atc 63

<210> 47 <211> 53 <212> DNA <213> artificial sequence <220> <223> Deep seq Enoyl_ACP_reductase_Rev

<400> 47 cctatcccct gtgtgccttg gcagtctcag gaaatggtgt atccgtccaa tcc 53

<210> 48 <211> 1629 <212> DNA <213> Phaeodactylum tricornutum <220> <223> delta 12 fatty acid desaturase (PHATR_25769)

<400> 48 ttttttttcc ctcggttcgg acttttttcc ctcgtcggtg gtccgtattg gatcaagtct 60 gtctgtgact tccgttagcg tcccatagtt tgttacactt ggctgtgaaa cgaatacgtt 120 cttggtctac ttactacaac gaagcaacca ccagcagcat gggtaaggga ggtcaacgag 180 ctgtagctcc caagagtgcc accagctcta ctggcagtgc tacccttagc caaagcaagg 240 aacaggtatg gacttcgtcg tacaaccctc tggcgaagga tgccccggag ctgccaacca 300 aaggccaaat caaggccgtc attccgaagg aatgtttcca acgctcagcc ttttggtcta 360 ccttctacct gatgcgcgat ctcgccatgg ctgccgcctt ttgctacgga acctcacagg 420 tcctctccac cgaccttccc caagacgcca cgctcattct gccctgggct ctcggctggg 480 gcgtctacgc cttttggatg ggaaccattc tcaccgggcc ttgggtagtt gcgcacgaat 540 gtggacacgg cgcttactcc gactcccaga cgttcaatga cgttgtcggc tttatcgtcc 600 accaagcttt gctcgtcccc tactttgcct ggcagtacac ccacgcgaaa caccaccgtc 660 gtaccaacca tctggtggac ggcgagtcgc acgtcccttc taccgccaag gataacggcc 720 tcgggccgca caacgagcga aactccttct acgccgcgtg gcacgaggcc atgggagacg 780 gcgcctttgc cgtctttcaa gtctggtcgc acttgttcgt cggctggcct ctctacttgg 840 ccggtctggc cagtaccgga aagcttgcgc acgaaggttg gtggctggaa gaacggaacg 900 cgattgcgga tcactttcga cccagctctc ccatgttccc cgccaagatc cgtgccaaga 960 ttgccctttc cagcgcgacg gaactcgccg tgctcgctgg actcttgtat gtcggtacac 1020 aggtcggaca ccttcccgtc ctgctgtggt actggggacc gtacaccttt gtcaacgctt 1080 ggcttgtact ctacacgtgg ctgcagcata cggacccgtc catcccgcac tacggtgaag 1140 gcgagtggac ctgggtcaag ggcgcgctct ctaccattga tcgagactac ggcatcttcg 1200 atttctttca ccacaccatc ggttccacgc acgtggtaca ccatttgttc cacgaaatgc 1260 cctggtacaa tgccggcatt gccacgcaaa aggtcaagga atttttggaa ccccagggct 1320 tgtacaatta cgatccgacc ccctggtaca aggccatgtg gcgcattgcc cggacctgtc 1380 actatgtgga gtcaaacgag ggtgtgcagt atttcaagag tatggaaaac gtgccgctga 1440 ctaaggatgt gcgaagcaaa gccgcatgag aaaaagtgcc accgacgcat aattttacaa 1500 tcctaccaac aagaccaaca ttatatggtt ttcgcttaaa agatagtttt ttctaccatc 1560 tgtgtagtcg gcacaactac gcgtcttagc ttggccgtca ccttgccggc tcgcgaggcc 1620 aaagttcgg 1629

<210> 49

<211> 5922 <212> DNA <213> artificial sequence <220> <223> pCLS19743 (TALEN Delta12 desaturase FAD2) <400> 49 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 Page 32 eolf-seql.txt catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat tacccatacg atgttccaga ttacgctatc gatatcgccg accccattcg 2400 ttcgcgcaca ccaagtcctg cccgcgagct tctgcccgga ccccaacccg atggggttca 2460 gccgactgca gatcgtgggg tgtctccgcc tgccggcggc cccctggatg gcttgccggc 2520 tcggcggacg atgtcccgga cccggctgcc atctccccct gccccctcac ctgcgttctc 2580 ggcgggcagc ttcagtgacc tgttacgtca gttcgatccg tcacttttta atacatcgct 2640 ttttgattca ttgcctccct tcggcgctca ccatacagag gctgccacag gcgagtggga 2700 tgaggtgcaa tcgggtctgc gggcagccga cgccccccca cccaccatgc gcgtggctgt 2760 cactgccgcg cggcccccgc gcgccaagcc ggcgccgcga cgacgtgctg cgcaaccctc 2820 cgacgcttcg ccggcggcgc aggtggatct acgcacgctc ggctacagcc agcagcaaca 2880 ggagaagatc aaaccgaagg ttcgttcgac agtggcgcag caccacgagg cactggtcgg 2940 ccacgggttt acacacgcgc acatcgttgc gttaagccaa cacccggcag cgttagggac 3000 cgtcgctgtc aagtatcagg acatgatcgc agcgttgcca gaggcgacac acgaagcgat 3060 cgttggcgtc ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc 3120 gggagagttg agaggtccac cgttacagtt ggacacaggc caacttctca agattgcaaa 3180 acgtggcggc gtgaccgcag tggaggcagt gcatgcatgg cgcaatgcac tgacgggtgc 3240 cccgctcaac ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca 3300 ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3360 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 3420 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 3480 cgccagccac gatggcggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3540 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg 3600 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 3660 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 3720 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg agcaggtggt 3780 ggccatcgcc agccacgatg gcggcaagca ggcgctggag acggtccagc ggctgttgcc 3840 ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg gtggccatcg ccagccacga 3900 tggcggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 3960 cggcttgacc ccggagcagg tggtggccat cgccagcaat attggtggca agcaggcgct 4020 ggagacggtg caggcgctgt tgccggtgct gtgccaggcc cacggcttga ccccggagca 4080 ggtggtggcc atcgccagca atattggtgg caagcaggcg ctggagacgg tgcaggcgct 4140 gttgccggtg ctgtgccagg cccacggctt gaccccccag caggtggtgg ccatcgccag 4200 Page 33 eolf-seql.txt caataatggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 4260 ggcccacggc ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca 4320 ggcgctggag acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4380 ccagcaggtg gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca 4440 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 4500 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 4560 gtgccaggcc cacggcttga ccccccagca ggtggtggcc atcgccagca ataatggtgg 4620 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4680 gaccccggag caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac 4740 ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc ttgacccctc agcaggtggt 4800 ggccatcgcc agcaatggcg gcggcaggcc ggcgctggag agcattgttg cccagttatc 4860 tcgccctgat ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct 4920 cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg ggggatccta tcagccgttc 4980 ccagctggtg aagtccgagc tggaggagaa gaaatccgag ttgaggcaca agctgaagta 5040 cgtgccccac gagtacatcg agctgatcga gatcgcccgg aacagcaccc aggaccgtat 5100 cctggagatg aaggtgatgg agttcttcat gaaggtgtac ggctacaggg gcaagcacct 5160 gggcggctcc aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg 5220 cgtgatcgtg gacaccaagg cctactccgg cggctacaac ctgcccatcg gccaggccga 5280 cgaaatgcag aggtacgtgg aggagaacca gaccaggaac aagcacatca accccaacga 5340 gtggtggaag gtgtacccct ccagcgtgac cgagttcaag ttcctgttcg tgtccggcca 5400 cttcaagggc aactacaagg cccagctgac caggctgaac cacatcacca actgcaacgg 5460 cgccgtgctg tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct 5520 gaccctggag gaggtgagga ggaagttcaa caacggcgag atcaacttcg cggccgactg 5580 ataactcgag cgatcctcta gacgagctcc tcgagcctgc agcagctgaa gctttaagat 5640 ccaatggcaa ggaccaagtg ctggaacttg ttttgcttta gcagatctag atcgagctac 5700 ctcgactttg gctgggacac tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa 5760 ctcaaccagg gacgtgcggc acaaatgggc atccttgctc tcatggtgca cgaacagttg 5820 ggagtctcta tccttcctta aaaatttaat tttcattagt tgcagtcact ccgctttggt 5880 ttcacagtca ggaataacac tagctcgtct tcatatcctg ca 5922

<210> 50

<211> 5940 <212> DNA <213> artificial sequence <220> <223> pCLS19747 (TALEN Delta12 desaturase FAD2) <400> 50 gggtacgttt aaacgtatta attaagacct agcatgtgag caaaaggcca gcaaaaggcc 60 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 120 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 180 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 240 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 300 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 360 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 420 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 480 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 540 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 600 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 660 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 720 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 780 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 840 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 900 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 960 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1020 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1080 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1140 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1200 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1260 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1320 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1380 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1440 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1500 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1560 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1620 Page 34 eolf-seql.txt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1680 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1740 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1800 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacaaact tggtaccata 1860 actagttcgg cgcgccaatc tcgcctattc atggtgtata aaagttcaac atccaaagct 1920 agaacttttg gaaagagaaa gaatgtccga atagggcacg gcgtgccgta ttgttggagt 1980 ggactagcag aaagtgagga aggcacagga tgagtttcct cgagacacat agcttcagcg 2040 tcgtgtaggc taggcagagg tgagttttct cgagacatac cttcagcgtc gtcttcactg 2100 tcacagtcaa ctgacagtaa tcgttgatcc ggagagattc aaaattcaat ctgtttggac 2160 ctggataaga cacaagagcg acatcctgac atgaacgccg taaacagcaa atcctggttg 2220 aacacgtatc cttttggggg cctccagcta cgacgctcgc cccagctggg gcttccttac 2280 tatacacagc gcatatttca cggttgccag aaccatgggc gatcctaaaa agaaacgtaa 2340 ggtcatcgat aaggagaccg ccgctgccaa gttcgagaga cagcacatgg acagcatcga 2400 tatcgccgac cccattcgtt cgcgcacacc aagtcctgcc cgcgagcttc tgcccggacc 2460 ccaacccgat ggggttcagc cgactgcaga tcgtggggtg tctccgcctg ccggcggccc 2520 cctggatggc ttgccggctc ggcggacgat gtcccggacc cggctgccat ctccccctgc 2580 cccctcacct gcgttctcgg cgggcagctt cagtgacctg ttacgtcagt tcgatccgtc 2640 actttttaat acatcgcttt ttgattcatt gcctcccttc ggcgctcacc atacagaggc 2700 tgccacaggc gagtgggatg aggtgcaatc gggtctgcgg gcagccgacg cccccccacc 2760 caccatgcgc gtggctgtca ctgccgcgcg gcccccgcgc gccaagccgg cgccgcgacg 2820 acgtgctgcg caaccctccg acgcttcgcc ggcggcgcag gtggatctac gcacgctcgg 2880 ctacagccag cagcaacagg agaagatcaa accgaaggtt cgttcgacag tggcgcagca 2940 ccacgaggca ctggtcggcc acgggtttac acacgcgcac atcgttgcgt taagccaaca 3000 cccggcagcg ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga 3060 ggcgacacac gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac gcgctctgga 3120 ggccttgctc acggtggcgg gagagttgag aggtccaccg ttacagttgg acacaggcca 3180 acttctcaag attgcaaaac gtggcggcgt gaccgcagtg gaggcagtgc atgcatggcg 3240 caatgcactg acgggtgccc cgctcaactt gaccccccag caggtggtgg ccatcgccag 3300 caataatggt ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca 3360 ggcccacggc ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca 3420 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 3480 ggagcaggtg gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca 3540 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccccagcagg tggtggccat 3600 cgccagcaat ggcggtggca agcaggcgct ggagacggtc cagcggctgt tgccggtgct 3660 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagca atattggtgg 3720 caagcaggcg ctggagacgg tgcaggcgct gttgccggtg ctgtgccagg cccacggctt 3780 gaccccggag caggtggtgg ccatcgccag caatattggt ggcaagcagg cgctggagac 3840 ggtgcaggcg ctgttgccgg tgctgtgcca ggcccacggc ttgacccccc agcaggtggt 3900 ggccatcgcc agcaataatg gtggcaagca ggcgctggag acggtccagc ggctgttgcc 3960 ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg gtggccatcg ccagcaataa 4020 tggtggcaag caggcgctgg agacggtcca gcggctgttg ccggtgctgt gccaggccca 4080 cggcttgacc ccccagcagg tggtggccat cgccagcaat aatggtggca agcaggcgct 4140 ggagacggtc cagcggctgt tgccggtgct gtgccaggcc cacggcttga ccccccagca 4200 ggtggtggcc atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct 4260 gttgccggtg ctgtgccagg cccacggctt gaccccggag caggtggtgg ccatcgccag 4320 caatattggt ggcaagcagg cgctggagac ggtgcaggcg ctgttgccgg tgctgtgcca 4380 ggcccacggc ttgacccccc agcaggtggt ggccatcgcc agcaataatg gtggcaagca 4440 ggcgctggag acggtccagc ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc 4500 ggagcaggtg gtggccatcg ccagccacga tggcggcaag caggcgctgg agacggtcca 4560 gcggctgttg ccggtgctgt gccaggccca cggcttgacc ccggagcagg tggtggccat 4620 cgccagcaat attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct 4680 gtgccaggcc cacggcttga ccccggagca ggtggtggcc atcgccagcc acgatggcgg 4740 caagcaggcg ctggagacgg tccagcggct gttgccggtg ctgtgccagg cccacggctt 4800 gacccctcag caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag 4860 cattgttgcc cagttatctc gccctgatcc ggcgttggcc gcgttgacca acgaccacct 4920 cgtcgccttg gcctgcctcg gcgggcgtcc tgcgctggat gcagtgaaaa agggattggg 4980 ggatcctatc agccgttccc agctggtgaa gtccgagctg gaggagaaga aatccgagtt 5040 gaggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga tcgcccggaa 5100 cagcacccag gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg 5160 ctacaggggc aagcacctgg gcggctccag gaagcccgac ggcgccatct acaccgtggg 5220 ctcccccatc gactacggcg tgatcgtgga caccaaggcc tactccggcg gctacaacct 5280 gcccatcggc caggccgacg aaatgcagag gtacgtggag gagaaccaga ccaggaacaa 5340 gcacatcaac cccaacgagt ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt 5400 cctgttcgtg tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca 5460 catcaccaac tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg gcggcgagat 5520 gatcaaggcc ggcaccctga ccctggagga ggtgaggagg aagttcaaca acggcgagat 5580 caacttcgcg gccgactgat aactcgagcg atcctctaga cgagctcctc gagcctgcag 5640 cagctgaagc tttaagatcc aatggcaagg accaagtgct ggaacttgtt ttgctttagc 5700 Page 35 eolf-seql.txt agatctagat cgagctacct cgactttggc tgggacactt tcagtgagga caagaagctt 5760 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 5820 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 5880 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc atatcctgca 5940

<210> 51 <211> 50 <212> DNA <213> artificial sequence

<220> <223> Delta12 desaturase target <400> 51 tagctcccaa gagtgccacc agctctactg gcagtgctac ccttagccaa 50

<210> 52 <211> 61 <212> DNA <213> artificial sequence

<220> <221> misc_feature <222> (31); (32); (33); (34); (35); (36); (37); (38); (39); (40) <223> Deep seq Delta12 desaturase_For ; n is a or c or t or g

<400> 52 ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnnn ctcgtcggtg gtccgtattg 60 g 61

<210> 53 <211> 50 <212> DNA <213> artificial sequence

<220> <223> Deep seqDelta12 desaturase_Rev

<400> 53 cctatcccct gtgtgccttg gcagtctcag tggcgagatc gcgcatcagg 50

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Claims (15)

1. A method for producing lipids comprising the step of:

(a) cultivating in a culture medium a diatom strain in which a gene selected from

elongase and UDP-glucose pyrophosphorylase genes has been inactivated by a rare

cutting endonuclease selected from a TALE-nuclease, a MBBBD-nuclease and a CRISPR/Cas9 nuclease;

(b) harvesting said cultivated diatom strain;

(c) extracting the lipids from said harvested diatoms.

2. The method according to claim 1, wherein said method comprises the preliminary steps of:

(i) Selecting a target sequence within the elongase or the UDP-glucose pyrophosphorylasegenes;

(ii) Engineering a TALE-nuclease, a MBBBD-nuclease and/or CRISPR/Cas9

nuclease to target said target sequence and inactivate said gene;

(iii) Introducing said TALE-nuclease, MBBBD-nuclease and/or CRISPR/Cas9

nuclease into said diatom;

(iv) Selecting the diatoms, in which said gene has been inactivated.

3. The method according to claim 2, wherein an exonuclease, such as Trex2, is further

introduced into the diatom to increase mutagenesis.

4. The method according to any one of claims 2 and 3, wherein said target sequence is selected within a sequence having at least 80% sequence identity with either SEQ ID

NO: 3 or SEQ ID NO: 14.

5. The method according to claim 2, wherein said target sequence has at least 80 %

sequence identity with either SEQ ID NO: 6 or SEQ ID NO: 17.

6. The method according to anyone of claims 2 to 5,furthercomprising introducing into the diatom a donor matrix comprising at least one homologous region to the target

sequence such that homologous recombination occurs between said donor matrix and said target sequence.

7. The method according to claim 6, wherein said donor matrix comprises a transgene encoding a gene involved in lipid metabolism.

8. The method according to any one of claims 1 to 7, wherein said diatom produces an

increased amount of shorter chain length fatty acids and/or fatty acid with a low

degree of saturation.

9. The method according to claim 8, wherein the increased amount of shorter chain length fatty acids and/or fatty acid with a low degree of saturation is suitable for

producing biofuel.

10. The method according to claim 9, further comprising the step of producing biofuel

from the extracted lipids.

11. The method according to any one of claims 1 to 8, wherein said lipids have high content of omega-3 fatty acids, such as docosahexaenoic acid (DHA) and

Eicosapentaenoic acid (EPA or icosapentaenoic acid).

12. The method according to claim 11, further comprising the step of transforming the extracted lipids into a cosmetic or a food product.

13. Lipids prepared according to the method of any one of claims 1 to 9.

14. A biofuel prepared according to claim 10.

15. A cosmetic or food product prepared according to claim 12.

Cellectis

Patent Attorneys for the Applicant/Nominated Person

SPRUSON&FERGUSON